Human CML cell lines induce the changes in gene expression patterns of OP9 stroma cells on the co-culture.

<p><b>(a)</b> K562 and MEG01, were analyzed for the expression of Notch ligands with FACS. Shaded areas indicate control staining. <b>(b)</b> OP9 cells were co-cultured with K562 or MEG01 cells. Eight days later, the CML cells were depleted, and the isolated OP9 cells were cultured (0.5×10⁵ cells) alone. The viable cell numbers were counted on day 4. <b>(c)</b> OP9 cells precultured alone, with K562, or MEG01 cells for 8 days were isolated, and the expression of indicated genes were analyzed with quantitative RT-PCR. The means and SEs of triplicate determination are shown. *<i>P</i> < 0.05. <b>(d)</b> OP9 cells precultured alone or with MEG01 cells for 8 days were isolated, and the expression of indicated molecules were analyzed with FACS.</p

OP9/L cells show characteristic changes in integrin receptor expression and altered integrin-mediated interaction with KOBA cells.

<p>(<b>a</b>) The expression of <i>Icam1</i> and <i>Vcam1</i> in OP9, OP9/L, and OP9/L was determined by quantitative RT-PCR. The means and SEs of triplicate determination are shown. *<i>P</i> < 0.01. (<b>b</b>) OP9, OP9/L, and OP9/L (+DAPT) cells were analyzed for the surface expression of indicated molecules with FACS. Shaded areas indicate control staining. (<b>c</b>) Expression of LFA-1 on KOBA cells was analyzed with two-color FACS using anti-αL and anti-β2 integrin antibodies (left). KOBA cells were cultured on OP9 or OP9/L cell monolayers in a matrigel invasion chamber for 16 hours, and the cells that transmigrated the filters were counted (right). The means and SEs of triplicate culture are shown. (<b>d</b>) Expression of VLA-4 on KOBA cells was analyzed with two-color FACS using anti-α4 and anti-β1 integrin antibodies (left). KOBA cells were cultured on OP9 or OP9/L cell monolayers for 6 hours, and the cells that stably attached onto the monolayers were counted (right). The means and SEs of triplicate culture are shown.</p

KOBA cells repress <i>Cdkn</i> expression and enhance proliferation of OP9 cells by Notch activation.

<p>(<b>a</b>) Expression of indicated Notch target genes was determined in OP9, OP9/L, OP9/P, and KOBA cells were determined using quantitative RT-PCR. The means and SEs of triplicate determination. *<i>P</i> < 0.01. (<b>b)</b> Cell surface expression of Notch receptors and ligands was analyzed in OP9 and KOBA cells, respectively, using FACS. Shaded areas indicate control staining (left). C2C12 myoblast cells were co-cultured with or without KOBA cells for 8 days, and <i>Notch1</i> transcripts in isolated C2C12 were determined by quantitative RT-PCR (right). The means and SEs of triplicate culture. *<i>P</i> < 0.05. (<b>c</b>) OP9, OP9/L, OP9/P, and OP9 cells cultured with coated Dll4-Ig protein were lysed and immunoblotted with anti-Hes1 and anti-actin antibodies (upper). Arrowhead indicate Hes1 signal at 31 kDa, and relative signal densities of Hes1 over actin are shown. OP9 cells were co-cultured with KOBA cells in the absence or continuous presence of varying concentrations of DAPT for 8 days, and the isolated OP9 cells were lysed and immunoblotted with anti-Hes1 antibody (lower). Arrowhead indicate Hes1 signal, and relative signal densities of Hes1 over Actin are shown. (<b>d</b>) Expression of indicated <i>Cdkn</i> genes was determined in OP9, OP9/L, and OP9/L induced in the presence of 15 μM DAPT. The means and SEs of triplicate determination. (<b>e</b>) OP9 cells (1 × 10⁴ cells) were cultured alone or with KOBA cells in the absence or presence of 15 μM DAPT for 8 days and the viable cell numbers were counted (left). OP9 cells (1 × 10⁴ cells) were also cultured with or without coated Dll4-Ig for 8 days, and the viable cell numbers were counted (right). The means and SEs of triplicate culture are shown.</p

KOBA-NL⁺, but barely KOBA-NL^–, cells induce the increase in EC numbers and altered expression of adherence molecules.

<p><b>(a)</b> KOBA cells were separated into NL⁺ and NL⁻ fractions with a cell-sorter. Normal unirradiated B6 mice were injected intravenously with the sorted KOBA cells (3 × 10³ cells/mouse), and the BM cells were analyzed with two-color FACS for the expression of indicated molecules 12 days later (left). The proportions of GFP⁺ KOBA cells, CD45⁻ PDGFRβ⁺ MCs, and CD45⁻ CD31⁺ ECs are indicated. The means and SEs of total KOBA, EC, and MC cell numbers in 3 recipients were assessed (right). <b>(b)</b> The surface expression of indicated molecules was analyzed with multi-color FACS in the CD45^– CD31⁺ (EC) and CD45^– PDGFRβ⁺ (MC) cell gates 12 days after KOBA inoculation. Shaded areas indicate control staining.</p

Modification of Gene Expression, Proliferation, and Function of OP9 Stroma Cells by Bcr-Abl-Expressing Leukemia Cells

<div><p>Expression of the <i>Bcr-Abl</i> fusion gene in hematopoietic progenitor cells (HPCs) results in the development of chronic myelogenous leukemia (CML), for which hematopoietic microenvironment plays an important role. We investigated the specific effects of an HPC line transduced with <i>Bcr-Abl</i>, KOBA, on BM-derived OP9 stroma cells. DNA microarray analysis revealed that OP9 cells co-cultured with KOBA cells (OP9/L) show diverse changes in the gene expression. OP9/L cells showed significant down-regulation of <i>Cdkn</i> genes and up-regulation of <i>Icam1</i>, leading to the increased proliferation capacity of OP9 cells and enhanced transmigration of leukemia cells through them. The effects were attributed to direct Notch activation of OP9 cells by KOBA cells. OP9/L cells also showed a markedly altered cytokine gene expression pattern, including a robust increase in a variety of proinflammatory genes and a decrease in hematopoietic cytokines such as <i>Cxcl12</i>, <i>Scf</i>, and <i>Angpt1</i>. Consequently, OP9/L cells promoted the proliferation of KOBA cells more efficiently than parental OP9 cells, whereas the activity supporting normal myelopoiesis was attenuated. In mice bearing KOBA leukemia, the characteristic genetic changes observed in OP9/L cells were reflected differentially in the endothelial cells (ECs) and mesenchymal stroma cells (MCs) of the BM. The ECs were markedly increased with Notch-target gene activation and decreased <i>Cdkn</i> expression, whereas the MCs showed a marked increase in proinflammatory gene expression and a profound decrease in hematopoietic genes. Human CML cell lines also induced essentially similar genetic changes in OP9 cells. Our results suggest that CML cells remodel the hematopoietic microenvironment by changing the gene expression patterns differentially in ECs and MCs of BM.</p></div

OP9/L cells show increased proinflammatory factor and decreased hematopoietic factor gene expression and enhance KOBA cell proliferation at the cost of supporting normal hematopoiesis.

<p>(<b>a</b>) OP9, OP9/L, and OP9/P cells were analyzed for the expression of indicated cytokine genes with quantitative RT-PCR. The means and SEs of triplicate determination are shown. *<i>P</i> < 0.01. (<b>b)</b> OP9, OP9/L, and OP9/L (+15 μM DAPT) cells were cultured for 3 days, and indicated cytokines in the culture supernatants were assessed with enzyme-linked immunosorbent assay. The means and SEs of triplicate culture are shown. *<i>P</i> < 0.05. (<b>c</b>) KOBA cells (1 × 10³ cells) were cultured with OP9 or OP9/L cell monolayers, and the viable cell numbers were counted on indicated days. The means and SEs of triplicate culture are shown. (<b>d</b>) Sorted lin⁻ BM cells (10⁵ cells) from normal B6 mice were cultured with OP9 or OP9/L cell monolayers for 5 days, and total recovered cell numbers were counted. An aliquot of the cells was analyzed with two-color FACS for indicated lineage markers, and the cell numbers of lin⁻ and mature hematopoietic cells were also assessed.</p

Leukemic invasion of KOBA cells induces distinctive effects on the gene expression and phenotypes of ECs and MCs in BM.

<p>(<b>a</b>) ECs and MCs were sorted from the BM of mice 14 days after KOBA cell inoculation, and the expression of indicated genes was determined with quantitative RT-PCR. The means and SEs from three mice are shown. <i>P</i> < 0.05. (<b>b</b>) The surface expression of indicated molecules was analyzed with multi-color FACS in the CD31⁺ (EC) and PDGFRβ⁺ (MC) cell gates before (black line) and 14 days after (red line) KOBA inoculation. Shaded areas indicate control staining.</p

<i>Bcr-Abl</i>^<i>+</i> KOBA leukemia cells repress the expression of Cdk inhibitors and enhance the proliferation of OP9 stroma cells.

<p>(<b>a</b>) KOP1 and KOBA were immunostained with anti-GFP antibody and DAPI. (<b>b</b>) KOP1 and KOBA cells (1 × 10⁵ cells) were co-cultured with OP9 cells. KOP1 cells crawled under OP9 cells, forming a cobblestone pattern, whereas KOBA cells were on top of them. Viable cell numbers of KOP1 and KOBA cells in the absence or presence of OP9 cells on day 4 are also shown. The means and standard errors (SEs) of triplicate culture. (<b>c</b>) Expression of indicated genes was determined in OP9, OP9/L, and OP9/P cells using quantitative RT-PCR. The means and SEs of triplicate determination. *<i>P</i> < 0.05 and **<i>P</i> < 0.01. (<b>d</b>) OP9 and OP9/L cells were immunostained with anti-Cip1 and anti-Kip2 along with DAPI. (<b>e</b>) OP9, OP9/L, and OP9/P cells (100 cells) were cultured alone, and the viable cell numbers were counted on day 8. NS, not significant.</p

The numbers of ECs are increased around KOBA leukemia cells in BM.

<p>(<b>a</b>) Normal unirradiated B6 mice were injected intravenously with KOBA cells (3 × 10³ cells/mouse), and the BM cells were analyzed with two-color FACS for the expression of indicated molecules before and 8 and 14 days after the inoculation (left). The proportions of GFP⁺ KOBA cells, CD45⁻ PDGFRβ⁺ MCs, and CD45⁻ CD31⁺ ECs are indicated. The means and SEs of total GFP⁻ CD45⁺, EC, and MC cell numbers in five recipients were assessed (right). *<i>P</i> < 0.01. (<b>b</b>) The femoral bones of before and 8 days after KOBA inoculation were immunostained with anti-GFP and anti-CD105 antibodies. Enlarged images of the boxed regions are indicated.</p