Examples of numerical chromosomal and telomeric alterations in GCTB stromal cells of male patients.

<p>Centromeric 3 (red), 4 (green), 6 (yellow) and X (light blue) signals show different levels of polysomy. Chromosome 4 trisomy in a cell disomic for the rest (3,6 and X) of the tested centrosomes (b) and chromosome 11 subtelomeric loss and tetrasomy in a cell of another case (c). Scale bar on <u>a</u> represents 5 μm; and 2.5 μm on <u>b</u> and <u>c</u>.</p

Immunoperoxidase (a-c) and immunofluorescence (d-e) detection in osteoclast rich areas and surrounding stroma (f and g), and clinicopathological correlations of Cx43 protein levels (h and i) in giant cell tumor of bone.

<p>Examples of tumors with moderate (a; score 3) and high (b; score 8) Cx43 levels in mononuclear cells. Strong Cx43 reaction in the preexisting osteoblast layer around bone spicules and in osteocytes (arrowhead) (c). A tumor nest and adjacent ring of reactive stroma are annotated separately for counting Cx43 (Alexa564, red) plaques (d; OC-osteoclasts). Higher power of (d) with osteoclasts encircled (e). Digital image segmentation highlights Cx43 plaques in orange for automated counting (f). Both the Cx43 positive area fraction (g) and the number of Cx43 positive plaques (h) are significantly reduced within tumor nests (p<0.01). Cx43 levels are also significantly reduced in aggressive vs active and in aggressive vs latent clinicoradiological tumor stages (i). Scale bar on (a) represents 30 μm on <u>a</u>, <u>b</u> and <u>c</u>; 80 μm on <u>d</u>, 30 μm on <u>e</u> and 15 μm on <u>f</u>.</p

Dye coupling test for measuring potential communication through gap junctions with flow cytometry.

<p>Scheme on the principle of the technique (a). Unlabelled cell are mixed with double dye labelled cells (orange) of the same kind at a ratio of 10:1 (a-b). Calcein (Mw:622 Da, green), after esterase cleavage becomes hydrophylic and can pass into adjacent cells through gap junctions, while the larger lypophylic DiI (red) is trapped within donor cell membranes (b). The proportion of single calcein labelled cells measured with flow cytometry (B+-, lower right box) indicating dye coupling, is significantly higher (p<0.001) in the control cell cultures (c) than in GCTB stromal cell cultures (d). Diagram showing the mean ± standard deviation of dye transfer in 3 independent experiments using stromal cells isolated from 3 patients (e). Scale bar on <u>b</u> represents 20 μm.</p

Detection of Cx43 levels and the subcellular distribution of Cx43 protein in primary GCTB stromal cell, bone marrow stromal (BM) cell and HDFa fibroblast cultures.

<p>Immunoperoxidase reaction reveals paranuclear clumps of Cx43 protein in the frequently binucleated neoplastic GCTB stromal cells (arrows) (a). Significantly less Cx43 is linked to cell membranes in GCTB stromal cells than in the control cells as tested using immunofluorescence (b-g; red) and digital image analysis (b). Arrowheads highlight characteristic localization of Cx43 in the endoplasmic reticulum-Golgi region in GCTB stromal cells (b and c, identical areas) and in cell membranes in HDFa fibroblasts (d and e, identical areas). Cx43 is dispersed throughout bone marrow stromal cells including cell membranes (f and g, identical areas). Vimentin reaction in b, d and f (green) highlights cell shape, while black and white images of identical areas (c, e and g) better reveal subcellular localization of Cx43. Cx43 transcript and protein levels detected using RT-PCR (i) and western blots (j), respectively. In western blots, control cells but not GCTB stromal cells show alkaline phosphatase sensitive bands (P1 and P2). Results in graphs show the mean ± standard deviation of three independent experiments. For blue nuclear staining hematoxylin (a) and Hoescht (b and d and f) were used. Scale bar on <u>a</u> represents 20 μm; and 10 μm on b, <u>c</u>, <u>d</u>, <u>e</u>, and 15 μm on <u>f</u> and <u>g</u>.</p

Kaplan-Meier plots of univariate Cox regression analysis of Cx43 immunoscores in giant cell tumor of bone.

<p>An increased hazard of progression (reduced PFS) is linked to scores 1–3 vs 4–8 (arrow) separating patient number around the median, N_score1-3 = 60 (48.8%), N_score4-8 = 63 (51.2%) (a). Log-rank test proves significantly reduced progression free survival (PFS) in tumors presenting low (scores 1–3) vs high (scores 4–8) Cx43 protein levels (b).</p

Immunofluorescence detection of Cx43 (red; a-e) along with CD163 (green; a-c) or α-smooth muscle actin (α-SMA, green; d) for defining Cx43 positive cell fractions (f and g) in giant cell tumor of bone.

<p>Cx43 positive mononuclear cells rarely co-localize with the monocyte/marcophage marker CD163 (a). Automated image segmentation (HistoQuant) highlights Cx43 in orange and CD163 in greeen in separate layers (b) and a 3^rd layer is used to count red Cx43 signals in green cells (arrowheads) (c). Cx43 signals (see double and single labeled insets) are more frequent in α-SMA deficient (upper panel), than in strongly α-SMA positive cells (d; lower panel, non-specific signals in red blood cells are encircled). Cx43 plaques are linked to mononuclear cells-some are partly engulfed by an osteoclasts (arrow)- and not directly to giant cells (e). Diagrams showing significant differences in Cx43 positive mononuclear cell fractions counted using HistoQuant image analysis (f and g). Cell nuclei are stained blue using Hoescht. Scale bar on <u>a</u> represents 30 μm on <u>a</u>, <u>b</u> and <u>d</u>; and 15 μm on <u>c</u> and <u>e</u>.</p