Detailed Characterization of Mesenchymal Stem/Stromal Cells from a Large Cohort of AML Patients Demonstrates a Definitive Link to Treatment Outcomes

Abstract

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.

Keywords: AML; BM-MSC; IL-10; characterization; chemoprotection; immunosuppression; risk-stratification.

Figure 1

Differentiation Capacity of BM-MSCs from HDs and AML Patients (A) Phase-contrast morphology of BM-MSCs from HD and AML patients. (B) Top: oil red O staining indicative of adipogenic differentiation potential of BM-MSCs from HD, LR-, IR-, and HR-AML subjects. Bottom: quantitative expression by qRT-PCR for the pan-adipogenesis transcription factors cEBPa and PPAR. (C) Top: alizarin red staining revealing the osteogenic differentiation capacity of BM-MSCs from HD, LR-, IR-, and HR-AML subjects. Bottom: quantitative expression by qRT-PCR for the pan-osteogenesis transcription factors ALPL, OSTERIX, and OSTEOPONTIN. Original magnification is indicated in microscopy images. n = 30 patients (10 LR-, 10 IR-, and 10 HR-AML) and n = 10 healthy donor controls. Error bars indicate the SEM values of the biological replicates. ^∗p < 0.05.

Figure 2

Functional Characterization of BM-MSCs from HDs and AML Patients (A) Proliferation measured as population doublings of BM-MSC cultures from LR-, IR-, and HR-BM-MSC compared with BM-MSCs from HD. Inset represents patient variability. n = 46 patients (16 LR-, 11 IR-, and 19 HR-AML) and n = 10 healthy donor controls. (B) Clonogenic capacity of BM-MSCs (number of CFU per 1,000 cells seeded) from HD and LR-, IR- and HR-AML subjects. p < 0.05. Right: representative crystal violet staining of BM-MSC-CFU/colonies from HD- and AML-derived BM-MSCs. n = 40 patients (13 LR-, 10 IR-, and 17 HR-AML) and n = 10 healthy donor controls. Error bars indicate the SEM values of the biological replicates.

Figure 3

In Vitro Immunosuppressive and Anti-Inflammatory Properties of BM-MSCs from HDs and AML Patients (A) Left: level of immunosuppression measured as percentage of CFSE⁺ non-proliferating cells. CSFE-labeled PBMCs were stimulated with PHA and then co-cultured with BM-MSCs from LR-, IR-, and HR-AML subjects for 5 days. BM-MSCs from HD and transformed BM-MSCs (tMSCs) were used as positive and negative controls, respectively. Right: the number of cycling (CSFE^mild/low) cells was determined by flow cytometry. Representative flow-cytometry histograms of CSFE-labeled PBMCs: R1, proliferating cells; R2 non-proliferating cells. n = 35 patients (11 LR-, 10 IR-, and 14 HR-AML) and n = 10 healthy donor controls. ^∗p < 0.05. Error bars indicate the SEM values of the biological replicates. (B) Concentration of the indicated cytokines in cell-culture supernatants determined by Luminex Multiplex assays. PBMCs from HD were co-cultured with BM-MSCs from 10 HD, 2 tMSCs, and 35 BM-MSCs from LR-, IR-, and HR-AML subjects. Error bars indicate the SEM values of the biological replicates. ^∗p < 0.05. (C) Similar protective effect of immunosuppressive BM-MSCs from HD and AML (LR-, IR- and HR-) individuals, but not tMSCs, to Ara-C/idarubicin cytotoxic treatment of HL60 AML cells. Error bars indicate the SEM values of the biological replicates. ^∗p < 0.05.

Figure 4

Five-Year OS Kaplan-Meier Curve Reveals that Higher Production of IL-10 by BM-MSCs Is Independently Associated with Shorter OS in AML Impact of patient's age (A), IL-10-secreted levels by BM-MSCs (B), white blood cell counts (WBC) (C), and percentage of blasts in the BM (D) on OS. n = 35 patients (11 LR-, 10 IR-, and 14 HR-AML) and n = 10 healthy donor controls.