Patient and healthy donor samples

Twelve healthy donors who were matched with 12 newly diagnosed pediatric ALL patients (9 pre-B-ALL, 3 T-ALL) treated at the Ankara City Children's Hospital. ALL patients with relapse and conditions affecting the bone marrow were excluded. Patients with relapsed ALL or conditions affecting the bone marrow were excluded from the study. Both patients and donors provided informed consent, and the study was approved by the local ethics committee (approval number: 2014-063). The ALL-BFM 2000 protocol was followed for the diagnosis, classification, and treatment of patients. The proportions of leukemic blasts were assessed microscopically using May-Grünwald Giemsa staining and by flow cytometry.

Isolation of mononuclear cells from bone marrow and peripheral bood

After plasma collection, bone marrow samples were diluted with PBS (BiochromGmbH, Merck Millipore, Germany) and layered onto Biocoll™ separating solution (1.077 g/ml, BiochromGmbH, Merck Millipore, Germany) in a 1:1 ratio. Centrifugation was performed at 2000 rpm for 20 min to isolate the mononuclear cells (MNCs). The buffy coat, containing MNCs, was collected and subjected to a second centrifugation step. MNCs (25 × 106) were seeded into 75-cm2 culture flasks following the protocol described in a previous study (Ok Bozkaya et al. 2015). Flasks were incubated at 37 °C in a humidified atmosphere with 5% CO₂, and the culture medium was refreshed every 3 days. The same procedure was used to isolate MNCs from peripheral blood. T and B leukemic cells were isolated from peripheral blood using the EasySep™ Human B and T Cell Isolation Kit (STEMCELL Technologies, Canada Inc.) for immunomagnetic negative selection. After isolation, cells were counted using a Beckman Coulter HmX-AL cell counter, and their characteristics were analyzed by flow cytometry.

MSCs harvest and culture

Mononuclear cells were cultured using DMEM-LG (Dulbecco's Modified Eagle Medium–Low glucose) supplemented with 10% fetal bovine serum (BiochromGmbH, Merck Millipore, Germany) and 1% penicillin/streptomycin (BiochromGmbH, Merck Millipore, Germany) for 96 h, with non-adherent cells being removed after 3 days. Adherent cells were allowed to proliferate, and once they reached 90% confluence, they were detached using a 0.25% trypsin/EDTA solution 0.02% in PBS, without Ca2 + , Mg2 + (Biochrom GmbH, Merck Millipore, Germany). The cells were passaged until the second passage (P2). During this passage, MSC differentiation tests and characterization assays were conducted.

Cell viability and proliferation capacity of MSCs

Each passage of MSCs was counted and reseeded at a density of 5 × 103 cells per cm2. Cell proliferation was monitored until passage 5. The number of population doublings (PDs) was calculated using the formula log10(N)/log10(2), where "N" is the ratio of cells harvested to cells seeded. Cell viability at each passage was assessed using Trypan Blue (Invitrogen) exclusion.

Characterization and Differentiation Assay of MSCs

MSCs were cultured in T25 flasks for 21 days to induce osteogenic and adipogenic differentiation. Once cells reached 100% confluence, they were washed with PBS, and MesenCult® Adipogenic and Osteogenic Stimulatory Supplements (STEMCELL Technologies, Canada) were added. After 3 weeks, cells were fixed in formalin and stained with Oil Red O (Sigma, USA) and Alizarin Red (Sigma-Aldrich) to assess adipogenic and osteogenic differentiation, respectively. Images were captured using an Olympus IX73 microscope with Olympus cellSens imaging software at 10 × magnification (NA:0.25/W.D:10 mm). The presence of adipocytes was determined using Oil Red O staining, an indicator of intracellular lipid accumulation. Lipid droplets were counted in five different fields, and the percentage of Oil Red O-positive MSCs was calculated by averaging the percentages. The analysis of osteogenic differentiation was performed by counting the bone nodules observed in five different fields under a light microscope (Olympus IX73). The counting was conducted independently by two different researchers to ensure accuracy and eliminate observer bias (Azadniv et al. 2020).

MSC surface markers were also analyzed using flow cytometry. Cells were stained with CD45-Alexa Flour® 488 (BioLegend, cat. no. 304019), CD34-FITC (BioLegend, cat. no. 343604) antibody staining and positive CD90-PE (BioLegend, cat. no. 328110), CD73-APC (BioLegend, cat. no.344006), and CD105-PE/Cy7 (BioLegend, cat. no. 323218) and analyzed on Navios-Beckman Coulter flow cytometer using Navios Software v1.2.

Co-culture experiments

ALL blasts matched with their corresponding MSCs from the same patient in co-culture experiments. Leukemic blasts isolated from patients using EasySep™ B and T Cell Isolation Kit were co-cultured with passage 2 MSCs in 12-well plates (Cellstar, Greiner Bio-One). Two approaches were used: (1) direct co-culture: 2.5 × 104 MSCs were cultured, and after 24 h, 2.5 × 104 blasts were added; (2) indirect co-culture: MSCs were seeded on the lower side of a Transwell system with 0.8-µm pore size (ThinCertTM Greiner Bio-One, Austria), and blasts were seeded on the upper side after 24 h. Following 96 h of co-culture, leukemic blasts were collected for flow cytometry analysis of apoptosis.

Annexin V/PI apoptosis assay

After 4 days of co-culture, leukemic blasts were stained with Annexin V-FITC and propidium iodide (BioLegend, cat. no. 640914) following the manufacturer’s protocol. The leukemic blasts were briefly rinsed in 500 µl Annexin binding buffer, and then 7 µl propidium iodide and 5 µl annexin-V-FITC were added to the cell suspension. The cells were incubated for 20 min and analyzed in flow cytometry (Navios Beckmann Coulter).

Immunocytochemistry

After co-culture, leukemic blasts were removed, and MSCs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. After the blocking step, cells were incubated overnight with primary antibody β-catenin (1:50, E247, Abcam). The next day, cells were stained with an HRP-conjugated anti-rabbit IgG secondary antibody (1:500, Abcam). Cells were visualized using DAB staining (3,3'-diaminobenzidine) (Abcam) and imaged with an Olympus BX43 microscope equipped with an Olympus DP21 digital camera using a 40 × objective (NA: 0.65/W.D: 0.6 mm) and 20 × objective (NA: 0.4/WD: 1.2 mm).

Statistical analysis

Immunohistochemical labeling was evaluated using the Image J IHC Profiler program. β-Catenin staining intensity was scored as follows: 0 (no staining), 1 + (weak staining), 2 + (moderate staining), and 3 + (strong staining). Five randomly selected slides from each group were analyzed. Statistical differences between groups were assessed using the Student’s t-test or one-way ANOVA. A p-value < 0.05 was considered statistically significant.