Spheres are resistant to replicative cell death after doxorubicin treatment.

<p>Colony forming ability after doxorubicin treatment was determined in KTOSA5 cells (* <i>p</i> = 0.008; ** <i>p</i><0.001) (A) and U2OS cells (♮ <i>p</i> = 0.008; ♮♮ <i>p</i><0.001) (B).</p

Primer sequences for the amplification of RT-PCR products from canine cell lines.

<p>Primer sequences for the amplification of RT-PCR products from canine cell lines.</p

Cancer stem cells are resistant to the cytotoxic effects of doxorubicin.

<p>Spheres were isolated from the canine osteosarcoma cell lines; KTOSA5 (A) and CSKOS (B), and the human osteosarcoma cell lines U2OS (C) and SAOS2 (D). Spheres and adherent cells were treated with the indicated doses of doxorubicin and cell viability was assayed 48 hr after treatment (* <i>p</i><0.005).</p

Gene expression analysis of canine osteosarcoma stem cells.

<p>A three-dimensional representation of a principle component analysis of expression microarray data derived from KTOSA5 adherent cells, spheres and mesenchymal stem cells (MSC) (A). Heirarchial clustering analysis of the expression data (cut off p-value of 0.005). Expression values are represented by colours: blue squares represent low-expressed genes, red squares represent high-expressed genes (B). Biological process analysis of differentially expressed genes in KTOSA5 spheres compared to adherent cells (FDR = 0.005) (C).</p

Top ten upregulated genes in KTOSA5 spheres compared to adherent cells (FDR = 0.005).

<p>Top ten upregulated genes in KTOSA5 spheres compared to adherent cells (FDR = 0.005).</p

Top biological functions of differentially expressed genes in KTOSA5 spheres compared to adherent cells (FDR = 0.005).

<p>Top biological functions of differentially expressed genes in KTOSA5 spheres compared to adherent cells (FDR = 0.005).</p

Cancer stem cells express a higher level of COX-2.

<p>Validation of microarray with qRT-PCR (A). Expression of COX-2 protein (B).</p

COX-2 inhibition suppresses sphere forming ability.

<p>KTOSA cells were pre-treated for 24 hr with the indicated doses of meloxicam prior to assaying for sphere forming ability (* <i>p</i><0.001) (A). KTOSA5 (B), CSKOS (C), U2OS (D) and SAOS2 (E) cells were pre-treated for 24 hr with the long-acting COX-2 inhibitor mavacoxib prior to assaying for sphere forming ability (* <i>p</i><0.001).</p

Cancer stem cells show an increased invasive potential <i>in vitro</i>.

<p>Invasive ability of KTOSA5 (A, B) and U2OS (C, D) spheres and adherent cells was analysed using a collagen based invasion assay. Invading cells were quantified by measuring the optical density at 560 nm. * <i>p</i><0.005.</p

COX-2 inhibition has no effect on cell viablilty or chemo-resistance of cancer stem cells.

<p>Dissociated KTOSA5 spheres and adherent cells (A), and CD34 sorted KTOSA5 cells (B) were treated with the indicated doses of meloxicam and cell viability was assayed 72 hr after treatment. KTOSA5 CD34- and CD34+ cells were treated with both indicated doses of meloxicam and 0.05 µM doxorubicin, cell viability was assayed 72 hr after treatment.</p