Page 120 - 2020_08-Haematologica-web

S. von Palffy et al.
leads to exhaustion of normal HSC.11 Therefore, inhibi- tion of the pro-inflammatory environment in the disease might have therapeutic potential.7 Hence, a better under- standing of the autocrine and paracrine signaling impor- tant for LSC survival and maintenance will not only be of great importance for characterizing disease biology and progression, but might also translate into the develop- ment of novel therapies targeting the LSC.
To identify key positive regulators of CML stem cells, we conducted a high-content cytokine screen on stem cell enriched primary chronic phase CML cells using an arrayed library of 313 unique human cytokines. This screen confirmed the positive regulatory effect of IL-3,12,13 IL-1a/b,8 GM-CSF,14 IL-6,15,16 and IFN-γ,17 cytokines previ- ously reported to expand primitive CML cells, and also identified several novel positive regulators. Among the novel positive regulators, we identified myostatin propeptide (MSTNpp), a muscle secreted protein not pre- viously implicated in the regulation of normal or malig- nant hematopoiesis, and demonstrate that MSTNpp pro- motes the growth and survival of both primitive CML and normal hematopoietic cells.
Methods
Patient samples and CD34 enrichment
Bone marrow and peripheral blood (PB) from untreated chron- ic phase CML patients, AML patients or blast crisis CML patients were obtained after written informed consent and in accordance with the Declaration of Helsinki. The Regional Ethics Committee (Dnr 2017/391) approved the study. All cellu- lar chronic phase CML samples included in the study are sum- marized in the Online Supplementary Table S1. For information on cell preparation, see the Online Supplementary Methods.
Cytokine screening
Chronic myeloid leukemia PB samples with high IL1RAP expression within the CD34+CD38low compartment, correlating with a high fraction of BCR-ABL1 positive cells,18,19 were select- ed for the screen (Online Supplementary Figure S1). IL1RAP expression in the CD34+CD38low cells was assessed by flow cytometry as previously described.19 In brief, 500 CD34+CD38low cells were sorted into pre-prepared cytokine plates containing 313 unique cytokines, and cell numbers were evaluated after seven days. For detailed information on cytokine library prepa- ration, antibody staining, cell sorting, experimental read out and screen validation, see the Online Supplementary Methods.
Cell cultures
In all cell culture experiments, cells were cultured in serum free StemSpanTM SFEM media (Stem Cell Technologies, Canada). Cell numbers were evaluated using CountBright Absolute Counting Beads (Thermo Fisher Scientific, USA) and Draq7 (BioStatus, UK) on a LSR Fortessa (BD Biosciences, USA).
Cell cultures of transgenic BCR-ABL mouse cells
Transgenic ScltTA/BCR-ABL mice20 were taken off tetracycline pellets to induce CML-like disease. Leukemic mice and age- matched wild-type B6.SJL mice were sacrificed 8-10 weeks post induction. Five thousand Lin–Sca-1+c-Kit+ (LSK) BM cells were sorted into individual wells of a 96-well plate containing 500 ng/mL of MSTNpp (catalog# 12012, lot# 0603297, Peprotech) or no cytokine control, and cell numbers were analyzed after seven days. For detailed information on cell isolation, antibody stain-
ing, sorting, and cell culture see the Online Supplementary Methods.
Colony-forming assays
CD34+ chronic phase CML cells, CD34+CD38low chronic phase CML cells, or equivalent normal BM cells, were cultured for a week with or without cytokine, prior to transfer to H4435 MethoCult (Stem Cell Technologies) according to the manufac- turer’s instructions. See the Online Supplementary Methods for details on readout and colony replating.
Co-culture experiments with primitive chronic myeloid leukemia cells and mesenchymal stromal cells
CD34+CD38low CML cells were sorted as described above, and plated onto mesenchymal stromal cells (MSC) established from primary CML BM cells in a 1:5 ratio. Details on MSC cultures and culture conditions can be found in the Online Supplementary Methods.
MSTNpp binding experiment
KU81221 cells were resuspended in PBS with 2% FBS. Two ug/mL of MSTNpp (catalog# 12012, lot# 0603297, Peprotech) or no cytokine was added together with a polyclonal rabbit IgG anti-MSTNpp antibody (Antibodies-online, USA), and stained on ice in the dark for 30 minutes (min). Cells were washed twice in PBS with 2% FBS, resuspended in goat anti-rabbit BV421 (BD Biosciences) and stained on ice in the dark for another 30 min. After a final wash, cells were analyzed on a LSRFortessa (BD Biosciences). Draq7 (BioStatus) was used as a viability marker.
MSTNpp ELISA
MSTNpp concentrations in PB plasma samples from 12 CML patients and four healthy donors, and BM plasma from five healthy donors, were diluted 1:20 and analyzed using Human Myostatin ELISA (prodomain specific) (BioVendor, Czech Republic) according to the manufacturer’s instructions.
MSTN reverse transcriptase-quantitative polymerase chain reaction
Relative MSTN expression in CD34+ cells, MNC and MSC from CML BM was assessed using reverse transcriptase quanti- tative polymerase chain reaction (RT-qPCR). For detailed methodology and assays used, see the Online Supplementary Methods.
RNA sequencing and gene set enrichment analysis
Regarding the gene set enrichment analysis (GSEA), CD34+CD38low (lowest 5% of CD38-expressing cells) cells from PB of five chronic phase CML patients were sorted into flat bot- tom 96-well plates with or without 500 ng/mL of MSTNpp (Peprotech), with cell numbers ranging from 2,000-11,000 cells, and incubated for 3 h or 24 h. For detailed information on RNA- extraction, cDNA synthesis, library preparation, sequencing and analysis pipeline, see the Online Supplementary Methods.
Phospho-flow cytometry
Cells were resuspended in serum free StemSpanTM SFEM medium (Stem Cell Technologies) and incubated for 3 h at 37°C 5% CO2, prior to 15 min stimulation with 500 ng/mL of MSTNpp (Peprotech) or 10 ng/mL of TGF-b1 (Peprotech). Following fixation with 1.6% paraformaldehyde for 10 min at room temperature and permeabilization with 90% ethanol at -80°C, cells were stained with phospho-specific antibodies. Details on staining and the antibodies used are described in the Online Supplementary Methods.
2096
haematologica | 2020; 105(8)