AraC and DNR activate MSCs, de novo which abrogates B-ALL target cell responses to chemotherapy agents in coculture. (A) Phalloidin, DAPI, or αSMA staining (original magnification ×40) of HS27a cells or healthy donor MSCs: at baseline or after exposure to the chemotherapy agents indicated. (B) Gene expression panel showing fold upregulation (compared with untreated) in HS27a cells after exposure to the chemotherapy agents AraC (i), DNR (ii), DEX (iii), and VCR (iv). (C) Cytokine bead assays for IL6 (i), IL8 (ii), and CCL2 (iii) (picograms per milliliter, y-axis) following exposure of HS27a cells to the chemotherapy agents indicated on the x-axis. All statistically significant comparisons (by unpaired Student t test) are as depicted: IL8, none vs AraC, P < .0001; IL8, none vs DNR, P = .002; IL8, none vs DEX, P = .001; and IL8, none vs VCR, P < .0001. CCL2, none vs AraC, P = .0169; CCL2, none vs DEX, P = .0166; and CCL2, none vs VCR, P = .0065. (D) MTS assays showing relative viability of SEM cells (y-axis) after treatment with AraC (i), DEX (ii), and VCR (iii) for 48 hours, after coculture with HS27a cells previously primed by chemotherapy before the treatment denoted on the x-axis. Data are shown relative to unprimed HS27a cells, set at 1. AraC-primed HS27a cells are highlighted throughout with a yellow arrow. All statistically significant comparisons (by unpaired Student t test) are as depicted: no pretreatment vs VCR, P = .041, and AraC vs VCR, P = .022 (i). No pretreatment vs VCR, P = .0087, and AraC vs VCR, P = .0087 (ii). No pretreatment vs VCR, P = .0006, AraC vs VCR, P = .0017 (iii). (iv) MTS assay showing relative viability of SEM cells (y-axis) after Transwell culture with primed HS27a cells as denoted on the x-axis. Data are relative to unprimed HS27a, set at 1. There are no statistically significant differences. All data are the mean ± SE of 3 independent experiments. *.01 < P ≤ .05; **.001 < P ≤ .01; ***.0001 < P ≤ .001; ****P ≤ .0001.