AraC, but not vincristine or nocodazole, increases ROS and mitochondrial transfer from MSCs to ALL cells in a murine xenograft model of ALL and stimulates formation of a nestin+niche. (A) Live imaging of tumor burden at days −3 and +3, with respect to the treatments given. (i) Images with color scale bar and control mice are shown. (ii) Quantification of the luciferase expression with region of intensity units on the y-axis and experimental conditions on the x-axis. PBS vs AraC, P = .0026; PBS vs VCR, P = .0023 and for PBS vs nocodazole, P = .0098. (Bi) αSMA staining of MSCs isolated and expanded from 1 control and 1 AraC-treated mouse (original magnification ×40). (ii) Phalloidin/DAPI staining of MSCs isolated and expanded from 3 control, AraC, VCR, and nocodazole-treated mice (original magnification ×20). ROS (mean fluorescence intensity [MFI], y-axis) (Ci) and mitochondrial mass (green MitoTracker MFI, y-axis) (ii) after treatment of mice bearing SEM xenografts with the agents indicated (x-axis). Cells were harvested from mice at day +3 after treatment with control, AraC, VCR, or nocodazole. All statistically significant comparisons (by unpaired Student t test) are as depicted. (i) ROS: PBS vs Ara-C, P = .0053, and (ii) MitoTacker mass: PBS vs AraC, P = .0014. (D) Immunohistochemistry of sections of representative whole femora from AraC- and VCR-treated mice. Femora are dual stained with human CD19 (brown) and murine nestin (pink). (i) In the AraC example, CD19⁺ cells are seen closely associating with a nestin⁺ niche, as indicated by the red boxes. (ii) In the VCR example, CD19⁺ cells (brown boxes) are not associated with nestin⁺ cells (pink boxes). (Ei) Mitochondrial mass (green MitoTracker MFI, y-axis) after treatment of mice bearing SEM xenografts with the agents indicated. SEM cells were harvested from mice treated with PBS, AraC, VCR, or AraC+VCR at day +3 after treatment with control, AraC, VCR, or AraC+VCR. (ii) Kaplan-Meier survival curves (n = 5 mice per group) for mice treated with PBS (blue), AraC (red), VCR (green), and AraC+VCR (purple). All statistically significant comparisons (by unpaired Student t test) are as depicted: PBS vs AraC, P < .0001, and AraC vs VCR, P < .0001. Survival of AraC+VCR-treated mice was significantly greater than that of each of the other 3 groups, by Mantel-Cox test: PBS vs AraC+VCR, P = .0253; AraC vs AraC+VCR, P = .0080; and VCR vs AraC+VCR .0305. **.001 < P ≤ .01; ****P ≤ .0001.