MiR-21 expression is mainly confined to the tumor stroma.

<p>A) <i>In situ</i> hybridization in FFPE samples of esophageal cancer localized miR-21 expression (blue signals) in cancer associated fibroblasts of the tumor stroma, but not in the tumor cells. Slides were counterstained with nuclear fast red. B) The adjacent normal squamous part on the same slide did not show miR-21 expression neither in the stroma nor in the squamous cells; C) Nuclear staining of U6 snRNA was used as an internal control; D) Negative control without probe. Bottom-left inserts show a 2 times bigger magnification of each image; E) Samples with high stromal component showed significantly higher levels of miR-21 expression than samples with a low stromal content. (P value  = 0.04 with unpaired T-test with Welch' s correction).</p

Histopathological criteria of the analyzed SCC patients.

<p>Histopathological criteria of the analyzed SCC patients.</p

Cell migration and invasion properties of KYSE-30 cells are increased upon co-culture with HGF-1.

<p>A) KYSE-30 cells juxtaposed with HGF-1 fibroblasts showed a significantly higher potential to migrate (P<0.0001) or invade (P = 0.0001) through the coated 8.0 μm pore-sized membrane as compared to the KYSE-30 cells not grown co-culture with normal fibroblasts. Images of representative microscopic pictures are shown on the left; B) 3 day old conditioned media from HGF-1 fibroblasts could significantly induce migration and invasion of KYSE-30 cells (P<0.0001 for both experiments). Images of representative microscopic pictures are shown on the left.</p

Effect of co-culturing normal fibroblasts with esophageal cancer cell lines on miR-21 expression levels.

<p>A) A schematic view of the co-culture system; B) MiR-21 expression in KYSE-30 cells and normal fibroblasts (HGF-1) after juxtaposition in a co-culture system; C) MiR-21 expression levels in KYSE-30 and HGF-1 cells after treatment with CM of HGF-1 and KYSE-30 cells, respectively; D) MiR-21 expression levels in FLO-1 adenocarcinoma and HGF-1 fibroblast cells after juxtaposition in a co-culture system; E) MiR-21 expression levels in FLO-1 and HGF-1 cells after treatment with CM of HGF-1 and FLO-1 cells, respectively. Data were normalized to expression after 24 h of co-culture. Each experiment was performed at least 2 times in triplicates.</p

Differential expression of miR-21 in 42 FFPE tumor samples in comparison with adjacent non-tumoral tissue.

<p>Quantitative RT-PCR analysis on FFPE samples of esophageal SCC patients shows higher levels of miR-21 in cancerous tissue as compared to the adjacent non-cancerous counterpart (P = 0.0007). MicroRNA levels are normalized to 5S rRNA. Values are presented as means ± standard deviation. The P value was determined with a 2-tailed Student' s t-test.</p

Expression analysis of 6 fibroblastic markers in normal fibroblasts co-cultured with esophageal cancer cell lines KYSE-30 (A), FLO-1 (B) and OE-33 (C).

<p><i>COL4A1</i> expression in fibroblasts was significantly decreased after 2 days of incubation with KYSE-30 (P = 0.03) and OE-33 (P = 0.04) cells and after 3 days of incubation with FLO-1 cells (P = 0.01). <i>TIMP3</i> expression was <i>s</i>ignificantly decreased after 2 and 3 days of incubation with KYSE-30 (P = 0.0002 and P>0.0001, respectively), after 2 and 3 days of incubation with FLO-1 cells (P = 0.0013 and P = 0.0006, respectively) and after 2 and 3 days of incubation with OE-33 cells (P = 0.0017 and P = 0.0004, respectively) when compared to the measurement 24 h after the start of the co-culture.</p