Page 60 - Haematologica Vol. 109 - July 2024

ARTICLE - Leukemia-induced interferon signature in stroma M.W.E. Smeets et al.
level of drug resistance, suggesting that the IFNα/b response may serve a different role in the pathobiology of BCP-ALL.
Methods
More details on the methods involved in this research are provided in the Online Supplementary Methods.
Mesenchymal stromal cells and primary B-cell precursor acute lymphoblastic leukemia cells
MSC were obtained from bone marrow aspirates taken from pediatric BCP-ALL patients and healthy donors as specified in Online Supplementary Table S1, using previously described procedures.17
Primary BCP-ALL cells were collected from bone marrow aspirates taken from children with newly diagnosed BCP-ALL (<18 years) which were left over after diagnostic procedures, and for which written informed consent was given by patients, parents, or guardians in line with institutional review board guidelines. The patients’ characteristics are presented in Online Supplementary Table S2. All samples were archived in liquid nitrogen and showed >90% leukemic blasts after having been thawed.
Healthy immune cells
Mononuclear cells were isolated from umbilical cord blood, with the approval of the ethics committee of Erasmus Univer- sity Medical Center and the scientific research committee of Franciscus Vlietland Hospital. Antibodies against B-, T-, and natural killer (NK)-cell, monocyte, granulocyte, and dendritic cell markers were purchased for immune cell characteriza- tion using flow cytometry (Cytoflex LX; Beckman Coulter).
Co-culture of leukemic cells, healthy immune cells and mesenchymal stromal cells
MSC (0.225x106) were seeded in six-well plates with 3 mL of medium and cultured for 24 hours. This medium was replaced with 3 mL medium containing 5.0x106 freshly thawed primary BCP-ALL cells or healthy immune cells, or 0.9x106 cells in the case of BCP-ALL cell lines. Cells were cultured for 40 hours before supernatants were taken to measure cytokine levels by Luminex multiplex immunoassay.18 In parallel, BCP- ALL and MSC were fractionated by flow sorting on a SH800S Cell Sorter (Sony, San Jose, CA, USA) and used as input for RNA sequencing. Both mono-cultured and co-cultured BCP- ALL cells and MSC were processed in the same way. Purity and viability checks were performed after sorting (Online Supplementary Figure S1, Online Supplementary Table S3).
measured after 40 hours of exposure to BCP-ALL cells by flow cytometry (Cytoflex S). Cells in the upper and lower compartments of the transwell were flow-sorted for RNA sequencing as mentioned above.
RNA sequencing
Total RNA (RNA integrity number >6.8) from sorted MSC and leukemic cells was used for ribonucleotide-depleted long-noncoding RNA-sequencing (NovaSeq 6000; Illumina, San Diego, CA, USA). EdgeR (version 3.28.1) was used to per- form differential gene expression analysis. Pathway analysis was performed using Pathvisio software.19
Lentiviral silencing
Primary BCP-ALL cells (875,000; 1.0x106 cells/mL) were cul- tured for 72 hours with and without IFN signature gene-si- lenced MSC (50,000). The viability of BCP-ALL cells was determined by flow cytometry.
Inhibition of interferon signaling
Inhibitors of IFNα/b (1.8 ng/mL), and IFNγ (R&D Systems; 3.0 ng/mL) were used in unstimulated co-cultures of MSC and BCP-ALL cells for 40 and 120 hours. Cell viability and the IFN-related gene expression signature were assessed with flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), respectively.
Drug exposure experiments
The sensitivity of leukemic cells to L-asparaginase, dauno- rubicin, and prednisolone in the presence of IFNα/b in- hibitors was evaluated by flow cytometry after 120 hours’ exposure. The drug concentration most discriminative for MSC-induced resistance was determined for each individual BCP-ALL sample prior to the inhibitor experiments (Online Supplementary Figure S2).
Quantitative reverse transcriptase polymerase chain reaction
RT-qPCR was performed using a QuantStudio 12K Flex Re- al-Time PCR System (Thermo Fisher Scientific) (Online Sup- plementary Methods).
Statistical analysis
One-way analysis of variance tests and one-sided (un)paired t tests were performed using GraphPad Prism. Correction for multiple testing was included when appropriate. A (paired EdgeR) P value <0.05 was considered statistically significant.
Results
B-cell precursor acute lymphoblastic leukemia cells induce an interferon-related gene signature in mesenchymal stromal cells
We performed RNA sequencing on sorted fractions of MSC
Direct cell-cell contact-mediated interferon response
Dye transfer from Vybrant DiI-labeled (Invitrogen) ALL cells to MSC was determined in direct co-cultures and indirect co-cultures using a transwell setup (0.4 μm pore-size; Corn- ing, New York, NY, USA). Accumulation of dye in MSC was
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