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

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

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

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 secrete pro-inflammatory cytokines when exposed to OS CSC whereas CSC are unaffected.

<p><b>(A)</b> Scheme of the experiments used to evaluate the secreted proteins showed in the following panels; <b>(B)</b> TGF- β1 and IL-6 ELISA assays of the supernatants collected from the different chambers of the transwell with MSC co-cultured with HOS-CSC. TGFβ1 is secreted exclusively by MSC co-coltured in the presence of stem-like cells whereaseIL-6 is secreated by MSC independently from the co-cultuing with MSC. However secreated IL-6 levels were increased in MSC when co-cultured with CSC. The amount of IL-6 detected in lanes 2 and 4 is most likely due to leakage from the upper chamber, as no amount could be detected in the control in lane 6, where no MSC were seeded; <b>(C)</b> IL-6 ELISA assay for the evaluation of the same phenomenon with MG63-CSC secretion. The trend ofMG63-CSC was the same observed for HOS-CSC cells.</p

Primers and probes.

<p>Gene symbol, name, function, accession number, primer sequences and selected probe are shown.</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 presence of HOS-CSC enhance the secretion of TGFβ1 from MSC that, in turn. autocrinally induce the activation of inflammatory pathways.

<p><b>(A)</b> Scheme of the experiments used to evaluate the secreted proteins showed in the following panels. The secreted protein (only the supernatant in the MSC upper compartment was) <b>(B)</b> and mRNA expression <b>(C)</b> of TGFβ1 in MSC when co-cultured with HOS-CSC, after 3 days (*p<0.05). The presence of HOS-CSC, increased the expression levels in MSC of two genes of the NF-kB pathway, RelA <b>(D</b> for protein and <b>E</b> for mRNA<b>)</b> and RelB <b>(F</b> for protein and <b>G</b> for mRNA<b>)</b>, as shown by direct NF-kB nuclear translocation (D and F panels) or transcriptional activation (E and G panels), *p<0.05. <b>(H)</b> When activated by the presence of HOS-CSC, MSC secreted higher amounts of IL-6, as assessed by ELISA assay, (*p<0.05).</p

Stromal cells enhance HOS-CSC migration via IL-6 and the expression of adhesion molecules.

<p><b>(A)</b> We assessed whether treatment with Tocilizumab affected HOS-CSC migration. MSC were treated with Tocilizumab [100 μg/mL] 2 hours prior CSC seeding. HOS spheres were trypsinized and single cells were let to migrate for 3 hours. As a control, medium only was added in the lower chambers, representative images; <b>(B)</b> Quantification of the migration assay shown in panel (A) (*p<0.05); <b>(C)</b> The expression levels of ICAM-1 were increased in HOS-CSC co-cultured with MSC. Data were obtained by Real Time PCR (*p<0.05) and confirmed by Western blot <b>(D,</b> representative image and densitometric quantification, T0 represents the protein expression level of parental cells from which CSC was obtained) (*p<0.05); <b>(E)</b> MSC were treated with 100 μg/mL Tocilizumab 24 hours prior CSC seeding. HOS-CSC spheres were then co-cultured by using tranwells with MSC and incubated for 6 hours. The RNA from CSC was then extracted and analyzed for the MET expression that shows a dramatic decrease in the absence of IL-6 (*p<0.05).</p