TC-derived soluble factors promoted neutrophil survival.

<p><b>A.</b> Neutrophils were cultured in a TC-CM or the control medium. At the indicated time points, live cells were evaluated by flow cytometry with FITC-conjugated annexin V and PI. Results were expressed as percentages of live cells (mean ± SEM of five independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05. <b>B.</b> Representative flow cytometric panels of dot plots of PMNs cultured in a TC-CM or control medium and stained with FITC-conjugated annexin V and propidium iodide (PI) at 24 (upper panels) and 48 (lower panels) hours. <b>C</b>. The GM-CSF release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results were expressed as mean ± SEM of seven independent experiments; ****p < 0.001; ***p < 0.005. <b>D-F.</b> Neutrophil survival in a TPC1-derived <b>(D-E)</b> or 8505c-derived <b>(F-G)</b> conditioned medium was evaluated in the presence of an anti-GM-CSF blocking antibody or the relative isotype control (10 μg/ml). At 24 hours, live cells were stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. <b>Figs E and G</b> illustrate representative flow cytometric panels of one out of five independent experiments. The results were expressed as mean ± SEM of five independent experiments; **p < 0.01; *p < 0.05.</p

The number of tumor-infiltrating neutrophils positively correlated with tumor size in human TC specimens.

<p><b>A and B.</b> Histological analysis of TC specimens stained with a monoclonal anti-CD66b antibody. Whole-tumor section density of CD66b⁺ neutrophils was scored at 200× magnification. Representative cases of papillary thyroid carcinomas with a high <b>(A)</b> and low <b>(B)</b> CD66b⁺ neutrophil count (arrows; hematoxylin counterstaining, 200×). <b>C.</b> CD66b⁺ neutrophil counts in tumor specimens were distributed according to the tumor size. The median value of tumor size served as a cutoff level. Results are shown as the median, the 25th and 75th percentiles (boxes), and 5th and 95th percentiles (whiskers); *p < 0.05, according to the two-tailed Mann–Whitney <i>U</i> test. <b>D.</b> Neutrophil density positively correlated with larger tumor size in TC patients (r = 0.43; p = 0.01; Pearson’s correlation test).</p

TC-derived soluble factors modified neutrophils’ kinetic properties.

<p>Neutrophils were stimulated with a TC-CM or the control medium for 18 hours. Within this time window, digital phase contrast images of 15 fields/well were captured every 15 minutes via a 20× objective in the Operetta high-content imaging system. PhenoLOGIC (PerkinElmer) was employed for image segmentation and to calculate the single-cell kinetic properties <b>(A)</b>, accumulated distance, <b>(B)</b> speed and straightness <b>(C)</b> in dedicated analysis sequence. The results were expressed as mean ± SEM of six independent experiments; ***p < 0.005; **p < 0.01; *p < 0.05. <b>D-E.</b> Timepoint analyses of time-dependent properties such as current step size (D) and current speed (E) illustrating dynamic changes of the behavior of neutrophils (in presence or absence of TC-CMs) in function of the elapsed time from treatment. The results were expressed as mean ± SEM of six independent experiments; ****p < 0.001.</p

TC-CMs induced morphological changes in neutrophils.

<p>Neutrophils were stimulated with a TC-CM or the control medium for 16 hours and then were imaged by means of an Operetta high-content imaging system at 20× magnification. The images were analyzed in the Harmony software with PhenoLOGIC (PerkinElmer) and a dedicated analysis sequence (morphological properties, method STAR) to evaluate cell area <b>(A)</b>, roundness <b>(B)</b>, radial mean <b>(C)</b>, symmetry <b>(D)</b>, width-to-length ratio <b>(E)</b>, and axial length <b>(F)</b>. The results were expressed as an increase or decrease compared to the control (mean ± SEM of five independent experiments); ***p < 0.005.</p

Correlation between the clinical variables and tumor-associated CD66b⁺ neutrophils in thyroid carcinoma.

<p>Correlation between the clinical variables and tumor-associated CD66b⁺ neutrophils in thyroid carcinoma.</p

TC-derived soluble factors induced activation of neutrophils.

<p><b>A–C.</b> Neutrophils were stimulated with a TC-CM or the control medium for 90 minutes, stained for neutrophil activation markers CD11b <b>(A)</b>, CD66b <b>(B)</b>, and CD62L <b>(C)</b> and subjected to cytofluorimetric analysis. The results were expressed as mean fluorescence intensity or percentages of positive cells gated on neutrophils (mean ± SEM of five independent experiments); ***p < 0.005, **p < 0.01, *p < 0.05. <b>D-F.</b> Representative flow cytometric panels with respect to the gating strategy of total cells <b>(D)</b>, singlets <b>(E)</b> and CD15+ CCR3- neutrophils <b>(F)</b>. <b>G-I.</b> Representative histograms illustrating mean fluorescence intensity (MFI) and cell counts for CD11b <b>(G)</b>, CD66b <b>(H)</b> and CD62L <b>(I)</b> for one out of five independent experiments. MFI = mean fluorescence intensity; FMO = fluorescence minus one.</p

TC-derived soluble mediators induced neutrophil chemotaxis.

<p><b>A.</b> Neutrophil chemotaxis toward TC-CM or the control medium was evaluated using 3 μm cell culture inserts in 96-well companion plates. Neutrophils (2.5 × 10⁶ cells/ml per 75 μl) were allowed to migrate (37°C, 60 minutes) toward a TC-CM or the control medium (235 μl per well). At the end of the incubation, the cells were centrifuged and resuspended in PBS (100 μl) and counted by flow cytometry. Data are expressed as migratory cells relative to the control (mean ± SEM of five independent experiments), **p < 0.01. <b>B.</b> The CXCL8/IL-8 release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results are expressed as mean ± SEM of seven independent experiments; ****p < 0.001. <b>C and D</b>. Chemotactic activity of neutrophils <i>via</i> a TPC1-derived <b>(C)</b> or 8505c-derived <b>(D)</b> conditioned medium was analyzed in the presence of blocking antibodies directed against CXCR1 and/or CXCR2 (10 μg/ml) or the related isotype control. Migratory neutrophils were counted by flow cytometry. The results are expressed as a percentage of isotype control (mean ± SEM of eight independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05.</p

TC-derived soluble factors induced the expression of proinflammatory and angiogenic factors by neutrophils.

<p>Human neutrophils were treated with a TC-CM or the control medium for 18 hours. At the end of the incubation, the cells were harvested and lysed for RNA isolation. <i>CXCL8/IL-8</i> <b>(A)</b>, <i>VEGF-A</i> <b>(B)</b>, and <i>TNF-α</i> <b>(C)</b> mRNA levels were evaluated by real-time PCR. The results are expressed as a fold change relative to the control (mean ± SEM of six independent experiments); **p < 0.01; *p < 0.05.</p