Figure 2.
The B-ALL–mediated MSC to CAF transition is contact-independent and mediated by transfer of mitochondrial elements. (A) Photomicrographs (magnification ×20) showing phalloidin/DAPI staining of HS27a MSCs after coculture with SEM (top) or SD-1 (bottom) conditioned media alongside the 18-gene panel showing fold upregulation (Y axis) compared with mean baseline of HS27a MSCs. (B) Cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) following exposure of HS27a MSCs to RPMI alone or SEM or SD-1 ALL conditioned media, as indicated on the X axis. Mean and standard error of mean from 3 independent experiments are shown. ∗.01 < P ≤ .05; ∗∗.001 < P ≤ .01; ∗∗∗∗P ≤ .0001. (C) Mitochondrial transfer to HS27a MSCs from SEM or SD-1 cells (X-axis) by MitoTracker assay (mean fluorescence intensity, Y axis). (D) Agarose gel images showing PCR products from human nuclear and mitochondrial DNA and murine nuclear and mitochondrial DNA, as indicated in each quadrant, after coculture of SEM cells with MS-5 murine stromal cells. Red boxes in lane 1 represent SEM alone, pink boxes in lane 2 MS-5 alone, and yellow boxes in lanes 3 to 5 flow-sorted MS-5 cells after coculture. (E) Photomicrographs (magnification ×20) showing phalloidin/DAPI staining of HS27a MSCs alone, and cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) production by HS27a cells: alone, after coculture with SD-1 cells, and after coculture with SD-1 cells depleted of mitochondrial nucleic acid by low-dose ethidium bromide. For the cytokine production, mean and standard error of mean from 3 independent experiments are shown. (F) Cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) following exposure of HS27a cells to SD-1-conditioned medium (blue bars) or RPMI-alone control (red bars) with (+) or without (−) 0.2-μm filtration.

The B-ALL–mediated MSC to CAF transition is contact-independent and mediated by transfer of mitochondrial elements. (A) Photomicrographs (magnification ×20) showing phalloidin/DAPI staining of HS27a MSCs after coculture with SEM (top) or SD-1 (bottom) conditioned media alongside the 18-gene panel showing fold upregulation (Y axis) compared with mean baseline of HS27a MSCs. (B) Cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) following exposure of HS27a MSCs to RPMI alone or SEM or SD-1 ALL conditioned media, as indicated on the X axis. Mean and standard error of mean from 3 independent experiments are shown. ∗.01 < P ≤ .05; ∗∗.001 < P ≤ .01; ∗∗∗∗P ≤ .0001. (C) Mitochondrial transfer to HS27a MSCs from SEM or SD-1 cells (X-axis) by MitoTracker assay (mean fluorescence intensity, Y axis). (D) Agarose gel images showing PCR products from human nuclear and mitochondrial DNA and murine nuclear and mitochondrial DNA, as indicated in each quadrant, after coculture of SEM cells with MS-5 murine stromal cells. Red boxes in lane 1 represent SEM alone, pink boxes in lane 2 MS-5 alone, and yellow boxes in lanes 3 to 5 flow-sorted MS-5 cells after coculture. (E) Photomicrographs (magnification ×20) showing phalloidin/DAPI staining of HS27a MSCs alone, and cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) production by HS27a cells: alone, after coculture with SD-1 cells, and after coculture with SD-1 cells depleted of mitochondrial nucleic acid by low-dose ethidium bromide. For the cytokine production, mean and standard error of mean from 3 independent experiments are shown. (F) Cytokine bead assays for IL-6, IL-8, and CCL2 (pg/mL, Y axis) following exposure of HS27a cells to SD-1-conditioned medium (blue bars) or RPMI-alone control (red bars) with (+) or without (−) 0.2-μm filtration.

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