Ct values of candidate HKG genes in CSC and native cell lines.

<p>Ct values of candidate HKG genes in CSC and native cell lines.</p

Transcriptome data from deep sequencing analysis of CSC and native cells of osteosarcoma.

<p>Transcriptome data from deep sequencing analysis of CSC and native cells of osteosarcoma.</p

Expression of genes involved in migration confirms a role for IL-6 in CSC metastatic process.

<p>Galectin-3 (**p<0.01) and STAT3 (*p<0.05) were evaluated in HOS-CSC spheres after 3 days of co-culturing with MSC, and exposed to mAb anti IL-6 by Real Time PCR.</p

MSC increase the proliferation of HOS-CSC.

<p>Homotypic cultures of CSC or co-cultures of CSC+MSC for 3 days were trypsinized, fixed and immunostained with anti-Ki67 antibody (green). Hoecst 33342(blue) was used to counterstain nuclei. <b>(A)</b> Representative image; <b>(B)</b> The percentage of Ki67 positive nuclei was quantified and expressed as ratio to the total number of cells (*p<0.05). Ki67 expression was increased in the presence of MSC as compared to CSC cultured alone. Images were acquired using the same exposure setting. Original magnification 20X. Figure shows representative images. <b>(C)</b> The number of colonies formed by HOS-CSC co-cultured with MSC were evaluated on soft-agar and quantified. MSC secreatome enhanced HOC-CSC tumorigenicity.</p

Gene expression of stem-cell markers confirmed the enhancement of stem-like features of CSC when are co-cultured with MSC in transwell.

<p><b>(A)</b> Scheme of the co-culture system of HOS-CSCwith MSC used in this study. The co-culturing was prolonged for 3 days; <b>(B)</b> Sox2, Oct4, Nanog, and CXCR4 expression evaluated by Real Time PCR in HOS-CSC spheres that were cultured alone or with MSC (in transwell). Gene expression of CSC was also compared to parental HOS adherent cells (T0) (*p<0.05); <b>(C)</b> CXCR4 was also evaluated by ELISA. (*p<0.05). Note that the levels of Oct4, Nanog, and CXCR4 markers in the spheres were significantly higher than the parental cell line. For Sox2, we saw a similar trend of increase in CSC spheres respect to parental cells, although this increase was more evident when CSC where co-cultured with MSC.</p

Validation of the identified housekeeping genes.

<p>The effect of gene expression normalization with optimal HKG was investigated on CSC and native cells from MG-63 and ACHN. (A) For the osteosarcoma cell line, the expression of the stem cell markers Nanog and cMyc were evaluated and normalized to the geometric mean of GAPDH and YWHAZ. ***p = 0.0007 for Nanog, ***p = 0.0003 for c-Myc. (B) For the ranal carcinoma cell line, the expression of Nanog and cMyc was normalized to the geometric mean of PPIA and HMBS. **p = 0.0043 for Nanog. (n = 6).</p

Model for the circuit between MSC and HOS-CSC.

<p>Recruitment of MSC to the tumor environment leads to enhanced proliferation of OS stem cells. The presence of CSC, in turn, leads to a consistent secretion of TGFβ1 that activates a stromal autocrine loop that might be responsible for the activation of NF-kB genes and IL-6 secretion by MSC. Indeed, neutralization of TGFβ1 reduces the amount of secreted IL-6. Pro-tumorigenic effects of MSC, via IL-6, including induction of HOS-CSC migration and sphere growth, can be counteracted also by IL-6 neutralizing antibody. The presence of MSC is also responsible for increased expression of adhesion molecules involved in intra- or extra-vasation and the expression of MET can be counteracted by IL-6 neutralization.</p

TGFβ1-dependent IL-6 secretion is responsible for increased CSC sphere formation.

<p><b>(A)</b> HOS-CSC were let to grow for 6 days in the presence of MSC; the latter cells were added every 24 hours with 100 μg/mL of Tocilizumab. Spheres were then photographed, counted, and quantified (scale bar, 500 μm). Representative figures and schematic representation of the performed assay; <b>(B)</b> Treatment with Tocilizumab significantly decreased the number of HOS-CSC spheres showed in panel A (*p<0.05); <b>(C)</b> IL-6 secretion by MSC is TGFβ1 dependent. MSC were treated with 1 μg/mL mAb αTGFβ1 2 hours prior CSC seeding, and re-added every 24 hours for the three days of co-culture. Supernatants from the MSC upper compartment were then collected and analyzed for IL-6 secretion by ELISA assay (*p<0.05).</p

The co-culturing of OS cells with MSC enhances the spherogenic potential and protein expression of stem-related markers in OS-CSC spehers.

<p><b>(A)</b> Scheme of the assays showed in this figure. <b>(B)</b> Representative pictures of the formed sphere of CSC in different conditions: with or w/o MSC in the upper chamber, see panel A (scale bar 500 μm, black arrows show the spheres with higher size); <b>(C)</b> spheres of CSC shown in panel A were counted and expressed as sphere forming efficiency (number of spheres formed / number of cells seeded × 100) (*p<0.05); <b>(D)</b> Average diameter of the counted spheres of CSC in panel C; <b>(E)</b> Stem cell markers of CSC spheres were evaluated by proteome expression profiler (left panel, image of the blotted membranes; right panel, densitometric evaluation of the same membranes and graphical representation of the obtained data); Oct4, Sox2 and Nanog have been highlighted on the membrane. Map of the blotted membrane and human pluripotent stem cell array coordinates are also shown.</p

Ranking of the stability of the expression of candidate reference genes by NormFinder, geNorm, and CV analyses in CSC and native cells from carcinoma and sarcoma tumors.

<p>Ranking of the stability of the expression of candidate reference genes by NormFinder, geNorm, and CV analyses in CSC and native cells from carcinoma and sarcoma tumors.</p