B-ALL cells directly stimulate MSCs to become CAFs. (A) Photomicrographs (magnification ×20) showing phalloidin/DAPI staining of HS27a MSCs after coculture with a series of ALL cell lines of different genetic subtypes, as indicated, alongside a gene expression panel showing fold upregulation (Y axis) of the 18-gene CAF panel compared with mean baseline of HS27a MSCs. (B) Cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) following coculture of HS27a MSCs with SEM or SD-1 ALL cells and controls of each cell alone, as indicated on the X axis. Mean and standard error of mean from 3 independent experiments are shown. P values for comparisons between HS27a + SEM and HS27a + SD-1 by unpaired t test are .0004 (IL-6), <.0001 (IL-8), and .0005 (CCL2). (C) Photomicrographs (magnification ×40) showing phalloidin/DAPI staining of 2 normal healthy donor (HD1, HD9) MSCs after coculture with 4 different primary patient ALL samples, 1-285, 1-456, 1-475, and 1-479. (D) IL-6, IL-8, and CCL2 (pg/mL, Y axis) secreted by HD1 and HD9 MSCs after coculture with the 4 individual primary patient ALL samples. (E) Mean fluorescent intensity (Y axis) of ROS after CellROX Green staining of the panel of ALL cell lines used in panel A. Mean and standard error of mean from 3 independent experiments are shown. (F) Representative sections of femur from NSG mice with established leukemia derived from SD-1 or SEM cells stained by CD19 (grayish brown) or nestin (pink).