Page 146 - Haematologica April 2020

L. Boutin et al.
sistant AML cells able to re-initiate the disease; these are likely to correspond to leukemia stem cells (LSC).
As a mirror of normal hematopoiesis, several studies reported a specific phenotype for LSC or leukemia-initiat- ing cells. Although this generated heterogeneous results,2 it was nevertheless reported that cells able to engraft de novo or after a secondary transplant were present in the CD34+CD38–CD123+ hematopoietic population.3-5 However, other studies showed that cells able to initiate leukemia concerned CD34–, CD33+ or CD13+ cells.6 Recently, Farge et al. demonstrated that chemoresistance was more related to a specific oxidative metabolism than to a level of progenitor/stem cell phenotype.7 Moreover, the high oxidative phosphorylation status is associated with elevated fatty acid oxidation and high expression of CD36, a fatty acid translocase recently identified as a marker of an LSC subpopulation.8
Considering hematopoietic neoplasms, it is now mandatory to also include environmental factors in order to understand their development, resistance and dissem- ination. In adults, hematopoiesis develops in the BM where a dialogue between hematopoietic stem /progeni- tor cells (HSPC) and the microenvironment, including mesenchymal stromal cells (MSC), extracellular matrix components and soluble factors,9,10 is critical for main- taining stem cell function and homeostasis. Such a pro- tective microenvironment has been reported to be impli- cated in the stemness and chemoresistance of leukemia blasts at the origin of the process of Environment Mediated-Drug Resistance (EM-DR), and specifically of Cell Adhesion Mediated-Drug Resistance (CAM-DR).8,11 Quiescence, as well as protection against environmental and drug aggressions, are major characteristics of stem cells also characterized by the Side Population (SP) phe- notype.12 We have previously shown that, whereas circu- lating HSPC from heathy donors (HD) do not exhibit a SP phenotype, this functionality can be induced after co-cul- ture with MSC in a VLA4- and CD44-dependent manner.13 Interestingly, this MSC-induced SP population is enriched in HSC, as shown by its engraftment in immunodeficient mice.13
In the present work, conducted on a cohort of 34 AML patients, we show that MSC activate ABC transporters in a subpopulation of primary blasts, resulting in the induc- tion of an SP phenotype. SP blasts are mostly quiescent and exhibit a low reactive oxygen species (ROS) tran- scriptional pathway compared to non-SP [Main Population (MP)] blasts. Furthermore, they are capable of effluxing chemotherapy agents in vitro in cultures as well as in vivo in patient-derived xenograft models treated by cytarabine, validating a higher chemoresistance of SP cells compared to their MP counterparts. Altogether, our results demonstrate that the stroma-induced SP function- ality is a new mechanism of CAM-DR for AML blasts.
Methods
Preparation of primary acute myeloid leukemia cells
Peripheral blood samples were collected at Percy (Clamart, France) and Saint Louis (Paris, France) hospitals after the informed consent of patients in accordance with the principles of the Declaration of Helsinki (IDRCB 2017-A02149-44, CPP 2017-juill.-14644 ND-1eravis, CNIL MR001). The patient cohort represents 34 patients with primary AML (Online Supplementary
Figure S1). Patients were untreated at the time of blood uptake. Blood mononuclear cells were isolated on density gradient (1.077g/mL) before freezing.13
Co-culture of primary acute myeloid leukemia cells on mesenchymal stromal cells
Primary AML mononuclear cells were plated at 2x105/cm2 in SynH (Abcell-Bio) supplemented with L-Glutamine, non-essen- tial amino acids and 10% fetal bovine serum for 3-4 days on confluent MSC isolated from the BM of either AML patients (n=9) or HD (n=5) (Online Supplementary Appendix). AML blasts were cultured without MSC feeders for controls. Transwell and neutralization experiments are described in the Online Supplementary Appendix.
In some experiments, mitoxantrone (50nM; Sigma Aldrich) was added to the co-culture for 24 hours (h) and cells were pre- treated or not with verapamil (50 μM) for 2 h before adding mitoxantrone and during mitoxantrone treatment.
At the end of co-cultures, non-adherent cells were flushed and stained with anti-CD45 antibody and annexin V (Invitrogen). Counting beads (CountBright, Life Technologies) were added to cell suspension to quantify cell populations by flow cytometry using Fortessa apparatus with Diva software (Becton Dickinson).
Side Population cell detection and characterization
Hoechst staining was performed as previously described13,14 (Online Supplementary Appendix). After Hoechst incubation, cells were placed on ice and stained with anti-CD45 antibody and a viability dye. Flow cytometry analysis was carried out on BD Fortessa apparatus. CD45 staining was use to gate on AML blasts and to avoid stromal contamination for SP analysis.
Drug efflux - to analyze drug efflux concomitantly with SP cell detection, mitoxantrone (90nM) was added to the cell suspen- sion during the last 30 minutes (min) of Hoechst staining.
ABC transporter functionality
Specific probes for ABCB1 (DioC2(3)), ABCC1 (CMFDA), and ABCG2 (Purpurin 18) were incubated during 30 min at 37°C after co-culture or during Hoechst staining. Cells were then stained with CD45 antibodies and with a viability dye (Online Supplementary Table S2).
Transcriptomic analysis
See the Online Supplementary Appendix.
Patient-derived xenograft model
Patient-derived xenografts (PDX) were achieved as previously described7,15 (Online Supplementary Appendix) under French Institutional Animal Care and Use (Committee of “Midi- Pyrénées” region-France) approval.
Statistical analysis
Raw data of each group were analyzed using R (3.3.3) and Rstudio (0.99.896) software. The packages used were stats, coin and multcomp for tests. Graphic representations of data were made for each group using Prism 6 or R (3.3.3) software (pack- age ggplot2). Statistical comparisons between groups on a single quantitative variable were run as follows: resampling tests were used for group versus group comparisons, pairing on AML donor levels or on AML MSC donor level. When multiple comparisons were used inside a single experiment, P-values were corrected using the Benjamini/Hochberg method. P=0.05 was considered statistically significant; tests were bilateral unless stated. Data are expressed as median with 25-75% interquartile intervals.
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