Survival analyses for PINP expression.

<p>In our set of samples, high PINP expression (PINP >50 %; n=41, 27 deaths in the group) compared to low expression (PINP 0-50%, n=71, 32 deaths) in OTSCC cells at the invasive front (>50%) correlated with increased overall deaths (A). In general overview, the increased expression of PINP in both OTSCC and stromal cells in the invasive areas of the tumor was observed in 76 patients (35 deaths in the group), whereas increased expression of PINP in superficial areas was observed in 36 patients (24 deaths). The higher expression of PINP in invasive areas associated with worse prognosis, p=0.004 (B).</p

CCL5 expression in OTSCC patient samples.

<p>CCL5 is mainly expressed by inflammatory cells in TME and some cancer cells in patient samples as shown by immunostaining with Pab CCL5, but only sparse CAFs are positive for CCL5 (A, B). CaDEC12 cells, the primary cultures of CAFs obtained from tongue cancer patients, express relative amount of CCL5 compared to NOFs, normal oral fibroblasts (C). Scale bar 100 µm.</p

BMMSCs inhibit cell proliferation in tongue cancer cells.

<p>Histological sections obtained from myoma disks were stained with polyclonal anti-Ki67 antibody. BMMSCs inhibit proliferation of HSC-3 and SAS cells in organotypic invasion model (A, B) as well as in cell culture assays (C), and shift HSC-3, but not dysplastic DOK cells towards a migratory phenotype with less cell-cell contacts (D, arrowheads). Normal gingival fibroblasts, GFs, promote wound closure more than BMMSCs potentially partly due to the increase in cell proliferation of cancer cells (E). Scale bar 50 µm.</p

BMMSCs enhance tongue cancer cell invasion in 3D organotypic model.

<p>Bone marrow-derived BMMSCs enhance the invasion of HSC-3 cells (A, B), but not dysplastic cells (E, F) in 3D myoma organotypic model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077692#B29" target="_blank">29</a>]. Minor increase, although not statistically significant, was seen in the invasion area of SAS (C,D). Histological sections obtained from myoma disks were stained with monoclonal pancytokeratin antibody (DAKO, clone AE1/AE3) and photographed at 100 x magnifications with a DMRB photo microscope connected to DFC 480 camera using QWin V3 software (Leica Microsystems). Scale bar 100 µm.</p

Signaling via CCL5/CCR5 is not critical in OTSCC invasion.

<p>The CCL5 receptor CCR5 is expressed in HSC-3 and SAS cells, but not in BMMSCs (A). rCCL5 can induce the expression of MMP9 in HSC-3 and SAS, but not in DOK cells (B). The silencing of the expression of CCR5 receptor in HSC-3 cells downregulates the gene expression about 70 % compared to control cells and blocks the signaling increasing the expression of MMP9 in cancer cells (C, D). CCR5 knockdown have minor or no effect on BMMSC enhanced invasion of HSC-3 cells in 3D organotypic model (E, F). </p

The expression of chemokine CCL5 is up-regulated in co-cultures of BMMSCs cells and OTSCC.

<p>Co-culture of OTSCC cells with BMMSCs results in the high expression of chemokine CCL5 in HSC-3 and SAS cells, but not in dysplastic DOK cells (A). Function-blocking monoclonal antibody against CCL5 (Mab CCL5) slightly inhibits BMMSC enhanced invasion area, but has no effect on the invasion depth, whereas the antibody against CXCL1 does not increase the inhibitory effect in OTSCC invasion (B, C).</p

The MTS absorbance at 490 nm is shown over 24, 48 and 72 h in the presence and absence of the 1.5 M short aptamer.

<p>The presence of the aptamer at 1 mM concentration was found to have no effect on the cell growth in comparison with the control.</p

Stern-Volmer plots for HSA titrated by short and long apatmers 37°C.

<p>Excitation wavelength 290 nm, [HSA]  =  6 µM. Excitation wavelength at 290 mM in a solution of sodium phosphate. Data is the mean of six values showing no greater standard deviation than 11%. The quenching effect is more considerable for long than short aptamer.</p

EIA and RIA assays.

<p>A: EIA (IIINTP indirect enzyme immunoassays) detecting N-terminal telopeptide from collagen type III degradation products at day 10 media change. Increasing absorbance means less collagen degradation product present. Hpa Ab (p = 0.03), 1.5 M Short (p = 0.007) and 1.5 M Long (p = 0.03) showed significant increase in absorbance compared to no inhibitor, suggesting they have inhibited the invasion of HSC-3 cells. B: The graph shows previous EIA values adjusted for negative control at day 10 media change. C: RIA (radioimmunoassay for type III collagen) detecting C-terminal telopeptide at day 10 media change. Increasing levels mean less collagen degradation product. Hpa Ab (p = 0.07), 1.5 M Short (p = 0.004) and 1.5 M Long (p = 0.02). D: RIA has confirmed the EIA assays showing significantly lower collagen degradation products than that for tissues without inhibitor added, indicating that they were successful inhibitors of invasion.</p

UV wavelength scan of HSA (left) and plot of PBS (right) titrated with 1.5 M short aptamer (A) and UV wavelength scan of HSA (left) and plot of PBS (right) titrated with 1.5 M long aptamer (B).

<p>UV wavelength scan of HSA (left) and plot of PBS (right) titrated with 1.5 M short aptamer (A) and UV wavelength scan of HSA (left) and plot of PBS (right) titrated with 1.5 M long aptamer (B).</p