Figure 2.

Mesenchymal stromal spheroids support primary leukemogenic B-ALL cells. (A) Primary sorted B-ALL CD10⁺CD19⁺ blasts were labeled with CTFR and co-cultured with HBM-MSC or ALL-MSC spheroids. After 24 h, spheroids were washed and analyzed by fluorescence microscopy and FACS. CTFR⁺ colonizer cell frequencies determined upon enzymatic digestion (n = 7). (B) CTFR-labeled primary B-ALL cells were treated with a CXCR4 inhibitor (AMD3100) 5 mM for 3 h and then co-cultured within PDLS. Upon 24 h, CTFR⁺ content was determined by FACS (n = 8). (C) Schematic representation of experimental design, 25,000 mononuclear cells from B-ALL patients were cultured in SF conditions, or co-cultured with HBM- or B-ALL-MSC monolayer (2D) or spheroids (3D). (D) Cell viability was analyzed by flow cytometry upon 48 h and absolute cell number was determined (n = 5) (left). Leukemic cells from independent experiments were harvested at 48 h of culture and transplanted into NSG mice. Human engraftment of hCD45⁺ was weekly monitored in PB by FACS and engraftment in BM was determined 6 weeks after transplantation (n = 4) (right). MSC, mesenchymal stromal cell; HBM, healthy bone marrow; B-ALL, B-cell acute lymphoblastic leukemia; CTFR, Cell Trace Far Red; FACS, Fluorescence-activated cell sorting; SF, stromal-free; PB, peripheral blood. *P < 0.05; **P < 0.01; ***P < 0.001, ***P < 0.0001. Error bars represent SD.