Phenotypes of tumor cell lines grown as single cell type tumor spheroids and fibroblast/tumor composite spheroid using the hanging drop method.

<p>Spheroids were grown from tumor cells alone (100%), or mixed with different proportions of wild type mouse embryonic fibroblasts. 50% and 10% refer to the amount of tumor cells in the composite spheroids. Spheroid morphology was assessed and photographed under a phase contrast microscope using a Nikon D3000 digital camera 4 and 6 days after seeding.</p

Spheroid size and interstitial fluid pressure.

<p>The size and interstitial fluid pressure (P_if) were determined for 4-day-old spheroids generated on agar plates (A-D) or by the hanging drop method (E and F). The spheroids are as indicated. (*p<0.05, **p<0.01, ***p<0.001).</p

Organization of tumor cells and stromal cells in composite spheroids.

<p>Composite spheroids of mouse fibroblasts and human tumor cells (as indicated) generated by the hanging drop method, were double-stained with antibodies towards human cytokeratin 7 or 18 (red) and mouse β1 integrin (green) at day 4 and day 6. Magnification: 10X, size bars = 100 µm.Tumor cell proliferation within tumor cell/fibroblast composite spheroids.</p

Cell surface expression of HS on wild-type fibroblasts, <i>Ext1^Gt/Gt</i> fibroblasts, A549 and H460 tumor cells.

<p>Representative flow cytometry fluorescence histograms of 10E4 antibody binding to A549 and H460 tumor cells (black profiles), wild-type fibroblasts (black profile) and <i>Ext1^Gt/Gt</i> fibroblasts (unfilled black curve). Controls represent cells treated only with the secondary antibody (gray profiles).</p

Morphology of single cell type spheroids and composite spheroids.

<p> Representative phase contrast images of multicellular spheroids generated by the hanging drop method after 4 days in culture. MEFs, mouse embryonic fibroblasts. Wild-type fibroblast spheroids, <i>Ext1^Gt/Gt</i> spheroids and composite spheroids, magnification 10X; tumor cell (A549, H460 and HeLa) spheroids, magnification 4X: all size bars = 100 µm.</p

Opposing effects of caspase inhibition on cardiac progenitor cell and differentiation markers expression.

<p>Relative mRNA levels of α-MHC (A), Nkx 2.5 (B), Isl1 (C), c-Kit (D) and Oct-4 (E) in EBs exposed to control vehicle or 10 μM Q-VD-OPH for the indicated times and then cultured for additional 7 days. * p<0.05 vs control, ** p<0.01 vs control.</p

Sublethal caspase activation enhances cardiac differentiation.

<p>(A) GFP expression levels in EBs exposed to vehicle or 100 nM STS +/- Q-VD-OPH (upper panels) and then cultured for additional 96 h (lower panels). Cardiac α-MHC promoter was driving GFP expression and differentiation was indicated as increase in GFP fluorescence compared to control. (B) In GFP negative zones of the EBs, new GFP positive cells were observed upon STS treatment. (C) Prospective long-term imaging of GFP expression in EBs exposed to control vehicle, 100 nM STS or 100 nM STS + Q-VD-OPH for 5 h and GFP expression was monitored during the following 72 h. (D) GFP expression level is expressed as the ratio between GFP levels at 72 h over GFP levels at 5 h, as monitored in individual, single cells. Relative mRNA expression levels of TnT (E), α-MHC (F), Nkx 2.5 (G) and Oct-4 (H) in EBs exposed to STS +/- Q-VD-OPH and collected for analysis after 3 and 7 days. Arrow indicates an individual, single cell after 5 h exposure to STS, which is then followed for 72 h,*p<0.05 vs control, ***p <0.001 vs control, ### p<0.001 vs STS.</p

Sublethal caspase activation promotes c-Kit + cardiac progenitors proliferation.

<p>Counterstaining for α-actinin and c-Kit in EBs exposed to 100 nM STS for 5 h and then cultured for additional 7 days. Area defined by the rectangle indicates clusters of c-Kit/α-actinin^low cells, and arrow indicates α-actinin^high cardiomyocytes. 7 days after the indicated treatments, EBs were pulsed for 30 minutes with EdU and then stained for α-actinin (green), c-Kit (red) and EdU (blue). Blue nuclei indicate proliferating cells. Bars represent 100 μm.</p

STS induces dose-dependent activation of caspases in differentiating EBs.

<p>(A and C) Enzymatic caspase 3 and 9 activity in response to stimulation with 1μM STS, 100 nM STS +/- Q-VD-OPH, and 10 μM Q-VD-OPH for 5 h. (B) Dynamic changes in mitochondrial potential detected in EBs as assayed by TMRE loss. (D and E) Caspase3 (active caspase 3 immunofluorescence) and caspase 9 (active caspase 9 immunofluorescence) activation in response to 1μM and 100 nM STS. Bars represent100 μm.</p

Caspase activation occurs mostly in non-differentiated cardiomyocytes.

<p>(A) Counterstaining of α-actinin (green), active caspase 3 (red) and nuclei (Dapi, blue) in EBs exposed to control vehicle or 100 nM STS +/- Q-VD-OPH for 5 h as indicated. Arrowheads indicate α-actinin^low cells positive for active caspase 3.</p