<p>Suplemmentary Material</p><p>Figures</p><p>Figure A – Schematic representation of metastatic and non-metastatic HNSCC patients groups as well the corresponding LN groups investigated in this study. Charts</p><p>Chart A – Methylated and non-methylated S100A4 primers sequences, methylation specific polymerase chain reactions (MS-PCR) amplification conditions, and expected product sizes in gel electrophoresis.</p><p>Fragments in gel Gene Primers Amplification conditions electrophoresis</p><p>Methylated Form 35 cycles at 95°C for 5 min., 95°C for 1 min, 56.5°C for 1 min. Forward, 5′-TATACGTTGTTGTTATAGTACG-3′ at 72°C for 30 sec., followed by a final extension at 72°C for 10 Methylated Form: min. Reverse, 5′-ACTTCCTACTCCCGAATACG-3′ 96 bp</p><p>S100A4 (first intron) Unmethylated Form 35 cycles at 95°C for 5 min., 95°C for 1 min, 56.5°C for 1 min. Unmethylated Forms: Forward, 5′-ATATGTTGTTGTTATAGTATGTG-3′ at 72°C for 30 sec., followed by a final extension at 72°C for 10 min. 94 bp Reward, 5′-CTTCCTACTCCCAAATACAC-3′ Chart B - Characteristics of anti-human primary antibodies and antigen retrieval conditions used in immunohistochemistry.</p><p>Primary Animal Antigen retrieval Endogenous blocking antibody species Manufacturer Clonality Dilution condition agents Specificity of primary antiboy</p><p>S100A4 Rabbit Sigma-Aldrich Polyclonal 1:300 Specificities of the binding of anti- Sections were submitted to ANXA2, MMP-9, endoglin, and CK1 Santa Cruz Blocking of endogenous ANXA2 Rabbit C-10 1:100 121 °C for 10 min in 10 mM antibodies were assessed by replacing Biotechnology peroxidase activity was citrate buffer, pH 6.0 by the primaries antibodies for a non- performed by reaction of Santa Cruz using a pressure boiler immune mouse immunoglobulin of the MMP-9 Mouse 2C3 1:100 the slides with 3% Biotechnology containing 10 mM sodium same isotype (product X0931, Dako). hydrogen peroxide (H2O2) citrate (pH = 6.0) for 10 min For anti-S100A4 and anti-ANXA2 solution for 20 min. Enzo Life at 125°C. The slides were antibodies, specificities were assessed Cytokeratin-1 Mouse 34βE12 1:100 Sections were submitted to Sciences then left in the pressure by using normal rabbit immunoglobulin endogenous avidin and boiler to cool down to 90°C fraction (product X0903, Dako) and biotin blocking by for 40 min. Sections were normal mouse serum (product X0910), incubation with 10% w/v submitted to enzymatic respectively. Positive controls were Dako bovine serum albumin and Endoglin Mouse SN6h 1:50 antigen retrieval with formalin-fixed, paraffin embedded Cytomation non-fat dry milk for 30 Proteinase K (Dako) in a tissues with known presence of the min each. humid chamber for 6min. target molecule according instruction of the manufacturer. Supplementary tables</p><p>Table A. Analysis between the S100A4 first intron methylation status and clinicopathological factors related to HNSCC.</p><p>S100A4 Methylation Status Variables p Methylated Non-methylated</p><p>Anatomical site</p><p>Anterior (n= 17) 7 (26.9%) 10 (38.5%) 0.375</p><p>Posterior (n= 35) 19 (73.1%) 16 (61.5%)</p><p>Tumor size</p><p>Small (n= 12) 7 (26.9%) 5 (19.2%) 0.510</p><p>Large (n= 40) 19 (73.1%) 21 (80.8%)</p><p>Locoregional metastasis</p><p>Absent (n= 26) 15 (57.7%) 11 (42.3%) 0.267</p><p>Present (n= 26) 11 (42.3%) 15 (57.7%)</p><p>WHO morphologic grade</p><p>I/II (n= 32) 15 (57.7%) 17 (65.4%) 0.569</p><p>III (n= 20 ) 11 (42.3%) 9 (34.6%)</p><p>Invasive front morphologic grade</p><p>4-12 (n= 25) 15 (57.7%) 10 (38.5%) 0.165</p><p>13-16 (n= 27) 11 (42.3%) 16 (61.5%)</p><p>Pattern of tissue invasion</p><p>Low invasiveness (n= 15) 9 (34.6%) 6 (23.1%) 0.358</p><p>High invasiveness (n= 37) 17 (65.4%) 20 (76.9%)</p><p>* Values bearing asterisks are statistically significant using Pearson´s chi-square and Fisher´s exact statistical tests. Table B – Analysis between immunohistochemical expression of S100A4, ANXA2, MMP9, and endoglin and clinicopathological factors related to HNSCC. </p><p>Immunohistochemical Tissue Expressions</p><p>Variables S100A4 ANXA2 MMP9 Endoglin</p><p> mean ± s.d. p mean ± s.d. p mean ± s.d. p mean ± s.d. p</p><p>Anatomical site</p><p>Anterior (n = 17) 46.70 (± 19.88) 0.158 85.59 (± 10.81) 0.476 38.40 (± 31.32) 0.732 9.38 (± 8.57) 0.232</p><p>Posterior (n = 35) 38.80 (± 18.02) 82.31 (± 18.51) 35.18 (± 31.67) 12.08 (± 7.04)</p><p>Tumor size</p><p>Small (n = 12) 37.98 (± 16.77) 0.480 80.60 (± 26.88) 0.617 36.89 (± 36.61) 0.935 11.61 (± 7.07) 0.833</p><p>Large (n = 40) 42.41 (± 19.49) 84.22 (± 11.95) 36.04 (± 30.02) 11.07 (± 7.83)</p><p>WHO morphologic grade</p><p>Well differentiated (n = 16) 37.24 (± 20.24) 0.461 87.05 (± 12.81) 0.176 44.97 (± 27.74) 0.397 11.54 (± 7.33) 0.951</p><p>Differentiated moderately (n =16 ) 45.63 (± 18.64) 78.86 (± 23.69) 32.57 (± 36.24) 11.39 (± 9.73)</p><p>Poorly differentiated (n = 20) 41.31 (± 18.00) 84.06 (± 10.78) 32.18 (± 29.83) 10.77 (± 6.13)</p><p>Invasive front morphologic grade</p><p>Low invasive pattern (n = 25) 39.66 (± 21.21) 0.531 83.28 (± 19.61) 0.667 40.74 (± 32.27) 0.322 10.83 (± 7.77) 0.743</p><p>High invasive pattern (n = 27) 42.98 (± 16.58) 83.48 (± 13.03) 32.06 (± 30.34) 11.53 (± 7.57) * Values bearing asterisks are statistically significant (p<0.05) using Mann-Whitney (for ANXA2 and MMP9) and Student´s t tests (for S100A4 and endoglin). Table C - Analysis of the S100A4 first intron methylation status and S100A4 protein expression in HNSCC and LN samples.</p><p>S100A4 S100A4 Groups p Methylation Status Expression (mean±s.d.)</p><p>Methylated 39.5 (± 22.9) 0.466 All HNSCC (n= 52) Unmethylated 43.3 (± 13.9)</p><p> metastatic HNSCC (n= 26) Methylated 47.5 (± 25.7) 0.894</p><p>Unmethylated 46.4 (± 15.8)</p><p> non-metastatic HNSCC (n= 26) Methylated 33.6 (± 19.4) 0.395</p><p>Unmethylated 39.2 (± 10.0)</p><p> mLN (n= 26) Methylated 44.4 (± 23.0) 0.903</p><p>Unmethylated 45.6 (± 18.0)</p><p> nmLN (n= 26) Methylated 52.1 (± 14.2) 0.786</p><p>Unmethylated 53.8 (± 15.0)</p><p> cLN (n= 26) Methylated 52.9 (± 15.3) 0.687</p><p>Unmethylated 49.1 (± 31.4)</p><p>* Mean values were performed using Mann-Whitney statistical test. mLN = metastasic lymph node; nmLN =non- metastasic lymph node; cLN = control lymph node. Table D – Correlation analysis among expression of S100A4, ANXA2, MMP9, and endoglin in metastatic and non-metastatic primary HNSCC and LN groups. </p><p>Metastatic Non-metastatic All HNSCC mLN nmLN cLN HNSCC HNSCC Variables r p r p r p r p r p r p</p><p>S100A4 vs. ANXA2 - 0.513 0.007* 0.308 0.126 0.023 0.873 -0.042 0.837 - 0.339 0.090 0.415 0.035*</p><p>S100A4 vs. MMP9 - 0.120 0.559 0.032 0.875 - 0.047 0.743 -0.373 0.061 - 0.229 0.260 - 0.142 0.488</p><p>S100A4 vs. endoglin - 0.248 0.222 - 0.163 0.428 0.087 0.541 0.222 0.277 - 0.122 0.554 - 0.130 0.526</p><p>ANXA2 vs. MMP9 0.023 0.912 0.197 0.335 0.138 0.328 0.158 0.441 0.128 0.535 0.319 0.113</p><p>ANXA2 vs. endoglin 0.408 0.039* - 0.060 0.769 0.126 0.372 0.167 0.416 - 0.250 0.218 - 0.012 0.955</p><p>MMP9 vs. endoglin 0.120 0.561 - 0.449 0.021* - 0.054 0.702 0.063 0.761 - 0.134 0.513 0.015 0.941</p><p>* Statistically significant results (p<0.05). r = correlation coefficient; mLN = metastasic lymph node; nmLN =non- metastasic lymph node; cLN = control lymph node.</p>