Figure 3.

PDLS promote stem cell-like phenotype, quiescence, and hypoxia in a subset of primary B-ALL cells. (A) Primary sorted B-ALL CD10⁺CD19⁺ blasts were labeled with CFSE and co-cultured with ALL-MSC to form PDLS. At 24 h, frequency of CFSE^hi was determined by FACS in the supernatants after spheroid removal (PDLS-out) and in the PDLS colonizer cells (PDLS-in) after several washes and enzymatic digestion (n = 5). (B) Primary B-ALL blasts were cultured in stromal-free (SF) conditions and co-cultured with ALL-MSC in monolayer (2D) and PDLS settings for 48 h and cell cycle status was evaluated by Ki-67 staining and DNA content by FACS (n = 5). (C) Side population cell contents are shown (n = 5). (D) ROS production was measured by FACS and ROSlow frequency was recorded (n = 5). (E) Hypoxia was investigated by HIF-1α expression (left), image-iT fluorescent hypoxia probe (middle) and pimonidazole incorporation (right) by FACS. Fluorescence microscopy of pimonidazole incorporation of PDLS is shown (n = 7). MSC, mesenchymal stromal cell; B-ALL, B-cell acute lymphoblastic leukemia; PDLS, patient-derived leukemia spheroids; CFSE, carboxifluorescein; FACS, fluorescence-activated cell sorting. *P < 0.05; **P < 0.01; ***P < 0.001, ***P < 0.0001. Error bars represent SD.