Suplemmentary Material

Figures

Figure A – Schematic representation of metastatic and non-metastatic HNSCC patients groups as well the corresponding LN groups investigated in this study. Charts

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.

Fragments in gel Gene Primers Amplification conditions electrophoresis

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

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.

Primary Animal Antigen retrieval Endogenous blocking antibody species Manufacturer Clonality Dilution condition agents Specificity of primary antiboy

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

Table A. Analysis between the S100A4 first intron methylation status and clinicopathological factors related to HNSCC.

S100A4 Methylation Status Variables p Methylated Non-methylated

Anatomical site

Anterior (n= 17) 7 (26.9%) 10 (38.5%) 0.375

Posterior (n= 35) 19 (73.1%) 16 (61.5%)

Tumor size

Small (n= 12) 7 (26.9%) 5 (19.2%) 0.510

Large (n= 40) 19 (73.1%) 21 (80.8%)

Locoregional metastasis

Absent (n= 26) 15 (57.7%) 11 (42.3%) 0.267

Present (n= 26) 11 (42.3%) 15 (57.7%)

WHO morphologic grade

I/II (n= 32) 15 (57.7%) 17 (65.4%) 0.569

III (n= 20 ) 11 (42.3%) 9 (34.6%)

Invasive front morphologic grade

4-12 (n= 25) 15 (57.7%) 10 (38.5%) 0.165

13-16 (n= 27) 11 (42.3%) 16 (61.5%)

Pattern of tissue invasion

Low invasiveness (n= 15) 9 (34.6%) 6 (23.1%) 0.358

High invasiveness (n= 37) 17 (65.4%) 20 (76.9%)

* 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.

Immunohistochemical Tissue Expressions

Variables S100A4 ANXA2 MMP9 Endoglin

mean ± s.d. p mean ± s.d. p mean ± s.d. p mean ± s.d. p

Anatomical site

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

Posterior (n = 35) 38.80 (± 18.02) 82.31 (± 18.51) 35.18 (± 31.67) 12.08 (± 7.04)

Tumor size

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

Large (n = 40) 42.41 (± 19.49) 84.22 (± 11.95) 36.04 (± 30.02) 11.07 (± 7.83)

WHO morphologic grade

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

Differentiated moderately (n =16 ) 45.63 (± 18.64) 78.86 (± 23.69) 32.57 (± 36.24) 11.39 (± 9.73)

Poorly differentiated (n = 20) 41.31 (± 18.00) 84.06 (± 10.78) 32.18 (± 29.83) 10.77 (± 6.13)

Invasive front morphologic grade

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

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.

S100A4 S100A4 Groups p Methylation Status Expression (mean±s.d.)

Methylated 39.5 (± 22.9) 0.466 All HNSCC (n= 52) Unmethylated 43.3 (± 13.9)

metastatic HNSCC (n= 26) Methylated 47.5 (± 25.7) 0.894

Unmethylated 46.4 (± 15.8)

non-metastatic HNSCC (n= 26) Methylated 33.6 (± 19.4) 0.395

mLN (n= 26) Methylated 44.4 (± 23.0) 0.903

nmLN (n= 26) Methylated 52.1 (± 14.2) 0.786

cLN (n= 26) Methylated 52.9 (± 15.3) 0.687

* 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.

Metastatic Non-metastatic All HNSCC mLN nmLN cLN HNSCC HNSCC Variables r p r p r p r p r p r 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*

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

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

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

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

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

* Statistically significant results (p<0.05). r = correlation coefficient; mLN = metastasic lymph node; nmLN =non- metastasic lymph node; cLN = control lymph node.