EXPRESSION OF TUMOR-DERIVED VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IS ASSOCIATED WITH OUTCOME IN

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1 DATA SUPPLEMENT EXPRESSION OF TUMOR-DERIVED VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IS ASSOCIATED WITH OUTCOME IN EARLY SQUAMOUS CELL CARCINOMA OF THE LUNG María J. Pajares et al. PATIENTS AND METHODS Validation of antibody specificities 1) Normal IgG (from the same species as the primary antibodies) or omission of the primary antibody was used as a negative control in the immunohistochemical technique. 2) Adsorption controls with the homologous VEGF and VEGFR1 antigens. Solutions containing specific antiserum (anti-vegfa and anti-vegfr1) were pre-incubated with the respective peptides (VEGFA: A-20 sc-152-p; and VEGFR1: C-17 sc-316-p, Santa Cruz Biotechnology) and applied to the sample instead of the primary antiserum. Subsequently, the immunohistochemistry procedure was performed. The adsorption control for VEGFR2 was not performed since the peptide was not commercially available. 3) Validation of the patterns of VEGF, VEGFR1, and VEGFR2 expression using different antibodies. For each marker, immunohistochemical techniques using different antibodies were performed in 30 randomly selected samples (10% of the tumors from the EUELC series). The validation antibodies were anti-vegfa (Ab-3 JH121, mouse monoclonal IgG, 1:100; Neomarkers,

2 Freemon, CA, USA), anti-vegfr1 (Ab-1, rabbit polyclonal IgG, 1:25; Neomarkers) and anti-vegfr2 (Ab-1, rabbit polyclonal IgG, 1:50; Neomarkers). The immunohistochemical method was identical to that previously described aside from the antigen retrieval. For VEGF, the antigen immunoreactivity was enhanced by microwaving the sections in a solution of ethylenediaminetetraacetic acid (1 mm EDTA, ph 8) for 2 x 10 min. For VEGFR1 and VEGFR2, antigen retrieval was performed in citrate buffer (10 mm, ph 6) for 2 x 15 min. 4) Western blot analysis. Proteins were extracted from tumor tissue blocks in the immediate vicinity of those studied by immunohistochemistry. Immunoblotting experiments were performed with the same antibodies used for immunostaining. Dilution of the primary antibodies was 1:200. To ensure equal loading and transfer of proteins, the membranes were subsequently probed with a polyclonal anti-actin antibody (1:500; Sigma-Aldrich). 5) sirna inhibition. Lung cancer cell lines were transfected with sirnas for VEGF, VEGFR1, and VEGFR2 (Dharmacon). The sequences of the sirnas were: VEGF: GAUCUCAUCAGGGUACUCCUU UGUCUUGCUCUAUCUUUCUUU VEGFR1: UAACAUGAAACCCAUUUGGUU UAACCAUACAACUUCCGGCUU, VEGFR2: UAGCUUGCAAGAGAUUUCCUU UAAAUGGAGAUCUGUAAUCUU Downregulation of the proteins was demonstrated by Western blot analysis and immunohistochemistry.

3 6) Validation of the immunohistochemical and quantification methods. Automatic immunohistochemical and semiquantification procedures were crossvalidated in 10% of the cases by an independent investigator (EB). RESULTS The vascular staining of VEGFR1 and 2 was used as an internal positive control (Figure S1). The specificity of antibodies used for immunohistochemistry was assessed using different technologies. Experiments with omission of the primary antibodies or incubation of samples with species and isotype-matched antibodies were performed in tumor samples in parallel with regular antibody incubation. These controls demonstrated no immunoreactivity (data not shown). Also, adsorption controls for VEGF and VEGFR1 rendered no staining (Figure S2). In order to verify the staining pattern of the antibodies, 10% of the tumor samples (30 patients) were stained with antibodies developed by another company (Neomarkers). This analysis revealed concordance greater than 80% for VEGF, VEGFR1, and VEGFR2 expression. Moreover, Western blot analysis was performed to confirm the molecular weight of the detected proteins. We found that the anti-vegf antibody recognized the three most common isoforms of VEGF (VEGF 121, VEGF 165 and VEGF 189 ) (Figure S3A), as previously reported 1. The antibody for VEGFR1 recognized a unique band of 180 kda (Figure S3B). In the case of VEGFR2, the antibody detected the unglycosylated form at 150 kda, the partially glycosylated form at 200 kda, and the fully glycosylated mature form at 230 kda (Figure S3C). Finally, sirna technology was used to validate our antibodies. VEGF, VEGFR1, and VEGFR2 were downregulated with specific sirnas and the expression of these proteins was

4 assessed by immunohistochemical and Western blot analysis. Low VEGF, VEGFR1, and VEGFR2 expression was demonstrated in the sirna transfected cells compared with the cells transfected with a scramble sirna, using both immunohistochemistry (Figure S4) and immunoblotting analysis (Figure S5). All of the controls strongly supported the specificity of the antibodies used in this study. Moreover, the immunohistochemical procedure and the semiquantification method were cross-validated in 10% of the cases. Concordance greater than 85% was found for all of the markers.

5 FIGURES Figure S1: Stromal (arrows) and endothelial cells (arrowheads) showed immunoreactivity for VEGFR1 and VEGFR2 in ADC (A, B) and SCC (C, D). The vascular staining of the VEGF receptors was used as an internal positive control for the immunohistochemical technique. Scale bar: 50 µm.

6 Figure S2: The specificity of the antibodies used for immunohistochemistry was demonstrated by adsorption controls. Immunoreactivity for VEGF (A) and VEGFR1 (C) was assessed in sections of tumor samples. Adsorption controls for VEGF and VEGFR1 were carried out in serial sections by preincubation (overnight at 4 ºC) of the primary antiserum at optimal dilution with the corresponding peptide: VEGF: A-20 sc-152-p (B), or VEGFR1: C-17 sc-316 P (D). Scale bar: 50 µm.

7 A. VEGF B. VEGFR1 C. VEGFR Figure S3: Western blot analysis in lung tumor samples demonstrated the specificity of the antibodies. (A) Western blotting for VEGF detected VEGF 121, VEGF 165 and VEGF 189 isoforms (B) The antibody for VEGFR1 recognized a unique band of 180 kda. (C) Western blot analysis for VEGFR2 showed 3 immunoreactive bands corresponding to the fully glycosylated mature form of VEGFR2 (230 kda), the partially glycosylated form (200 kda) and the unglycosylated form (150 kda).

8 A B C D E F Figure S4: sirna inhibition of VEGF, VEGFR1 and VEGFR2 was demonstrated by immunohistochemistry. VEGF (A-B), VEGFR1 (C-D), and VEGFR2 (E-F) expression was evaluated in NSCLC cells transfected with sirna targeting VEGF (NCI-H157), VEGFR1 (NCI-H727) and VEGFR2 (NCI-H358), respectively. Scale bar: 50 µm.

9 A. VEGF scr sirna B. VEGFR2 scr sirna Figure S5: Western blot analysis using the specific antibodies showed the downregulation of VEGF (A) and VEGFR2 (B) in sirna treated NSCLC cells (NCI-H157 and NCI-H358 respectively). Scr: scramble.

10 TABLES Table S1: Biomarker risk factors of disease progression for the EUELC series DF PD Median Cutoff No. of Patients % No. of Patients % Hazard Ratio* 95% CI P VEGF > to VEGFR > to VEGFR > to VSS > to MVD > to * Fine and Gray hazard ratio and P value for disease progression risk adjusted by pt, pn and smoking duration. CI, confidence interval; DF, disease-free; HR, hazard ratio; MVD, microvessel density; PD, progression disease; VSS, VEGF signaling score.

11 Table S2: Biomarker risk factors of disease progression for Stage I SCC patients of the EUELC series DF PD Median Cutoff No. of Patients % No. of Patients % Hazard Ratio* 95% CI P VEGF > to VEGFR > to VEGFR > to VSS > to MVD > to *Fine and Gray hazard ratio and P value for disease progression risk adjusted by smoking duration. CI: confidence interval; DF: disease-free; HR: hazard ratio; MVD: microvessel density; PD: progression disease; VSS: VEGF signaling score.

12 Table S3: Biomarker risk factors of disease progression for Stage I SCC patients of the MD Anderson series. Multivariate analysis of prognostic factors. DF PD Median Cutoff No. of Patients % No. of Patients % Hazard Ratio* 95% CI P VEGF > to VEGFR > to VEGFR > to VSS > to * Fine and Gray hazard ratio and P value for disease progression risk adjusted by pn, pt, sex, age and smoking status. CI: confidence interval; DF: disease-free; HR: hazard ratio; MVD: microvessel density; PD: progression disease; VSS: VEGF signaling score.

13 REFERENCES 1. Catena R, Larzabal L, Larrayoz M, et al: VEGF 121 b and VEGF 165 b are weakly angiogenic isoforms of VEGF-A. Molecular cancer 9: , 2010

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