American Society of Hematology

Figure 5

$Figure 5. FRCs support the survival of Eμ-Myc lymphoma cells in vitro. (A) Analysis of single-cell suspensions prepared from spleen and LNs by collagenase digestion (n = 4 mice), followed by in vitro culture for 3 to 7 days (n = 4 experiments). Cells were stained with anti-gp38, anti-CD45, and anti–VCAM-1. On the right, quantitative RT-PCR analysis of cell cultures that were further depleted of CD11b+ macrophages. CCL19 and CCL21 gene expression relative to glyceraldehyde-3-phosphate dehydrogenase (n = 2 experiments). (B) Confocal microscopy analysis of in vitro cultured gp38+ stromal cells. Cells were stained with anti-gp38 (green) and anti-α smooth muscle actin (SMA); (red) antibodies. Anti-CD45 antibody (red) was used to detect lymphoma cells in close contact to gp38+ stromal cells (right). (Bottom) A reconstructed side view along the contact zone between gp38+ layer and tightly adherent tumor cells (n = 2 experiments). (Left) Scale bar represents 20 μm. (Middle and right) Scale bars represent 50 μm. (C) Forward scatter (FSC) and side scatter (SSC) profiles of freshly isolated Eμ-Myc lymphoma cells cocultured for 20 hours with or without stromal cells containing approximately 60% gp38+/CD45− cells. (D) Lymphoma cells were cocultured with the gp38+/CD45− fraction, or the gp38− stroma cell fraction. For inhibition of hedgehog signaling, cyclopamine (20μM) was included for 20-22 hours (right panel). Lymphoma cell apoptosis was assessed by annexin V and 7-AAD staining. Results are given as x-fold cell death relative to lymphoma cells grown in the presence of stromal cells, set arbitrarily to 1 (n = 7 independent experiments). **P ≤ .01. n.s. indicates not significant. (E) RT-PCR analysis for Ihh and Shh. RNA derived from gp38+/CD45− enriched stromal cells and stomach (left panel). The hedgehog protein Ihh is located within the T-cell zone of splenic white pulp. Formalin-fixed spleen sections from Wt animals were stained with goat anti–mouse CD3 to detect T cells (red), and a rabbit anti–mouse Ihh antibody (dark blue) to stain stromal cells. (Right) Magnification of boxed inset on the left. Scale bars represent 100 μm.$

FRCs support the survival of Eμ-Myc lymphoma cells in vitro. (A) Analysis of single-cell suspensions prepared from spleen and LNs by collagenase digestion (n = 4 mice), followed by in vitro culture for 3 to 7 days (n = 4 experiments). Cells were stained with anti-gp38, anti-CD45, and anti–VCAM-1. On the right, quantitative RT-PCR analysis of cell cultures that were further depleted of CD11b⁺ macrophages. CCL19 and CCL21 gene expression relative to glyceraldehyde-3-phosphate dehydrogenase (n = 2 experiments). (B) Confocal microscopy analysis of in vitro cultured gp38⁺ stromal cells. Cells were stained with anti-gp38 (green) and anti-α smooth muscle actin (SMA); (red) antibodies. Anti-CD45 antibody (red) was used to detect lymphoma cells in close contact to gp38⁺ stromal cells (right). (Bottom) A reconstructed side view along the contact zone between gp38⁺ layer and tightly adherent tumor cells (n = 2 experiments). (Left) Scale bar represents 20 μm. (Middle and right) Scale bars represent 50 μm. (C) Forward scatter (FSC) and side scatter (SSC) profiles of freshly isolated Eμ-Myc lymphoma cells cocultured for 20 hours with or without stromal cells containing approximately 60% gp38⁺/CD45⁻ cells. (D) Lymphoma cells were cocultured with the gp38⁺/CD45⁻ fraction, or the gp38⁻ stroma cell fraction. For inhibition of hedgehog signaling, cyclopamine (20μM) was included for 20-22 hours (right panel). Lymphoma cell apoptosis was assessed by annexin V and 7-AAD staining. Results are given as x-fold cell death relative to lymphoma cells grown in the presence of stromal cells, set arbitrarily to 1 (n = 7 independent experiments). **P ≤ .01. n.s. indicates not significant. (E) RT-PCR analysis for Ihh and Shh. RNA derived from gp38⁺/CD45⁻ enriched stromal cells and stomach (left panel). The hedgehog protein Ihh is located within the T-cell zone of splenic white pulp. Formalin-fixed spleen sections from Wt animals were stained with goat anti–mouse CD3 to detect T cells (red), and a rabbit anti–mouse Ihh antibody (dark blue) to stain stromal cells. (Right) Magnification of boxed inset on the left. Scale bars represent 100 μm.

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