Cyto/Chemokines Quantification in Cell Culture Media

SUPPLEMENTARY DATA

MATERIALS AND METHODS

Cyto/chemokines quantification in cell culture media

The plates were assayed for a total of human 51 targets: EGF, FGF basic, GCSF, GM-CSF, GRO alpha, HGF, IFN alpha, IFN beta, IFN gamma, IL-1 alpha, IL-1 beta, IL-1 ra, IL-2, IL-4, IL-5, IL-6,

IL-7, IL-8, IL-10, IL-12 (p40), IL-12, (p70), IL-13, IL-15, IL-17A, IL-17F, ITAC, leptin, MCP-1,

MCP-3, MCSF, MIG, MIP-1alpha, MIP-1 beta, MIP-3 alpha, PAI-1, PDGFBB, RANTES, SAA, sCD40 ligand, TGF beta, TGF alpha, TNF alpha, TNF beta, VEGF, MIF, SDF-1, sE-selectin,

MMP-2, MMP-3, MMP-7, and MMP-9. Beads were washed and re-suspended with 50 l of sample or standard. Standards and samples were incubated for 60 min with beads, which were then read in a Luminex instrument (Bio-Rad) that was calibrated prior to use using Bio-Rad calibration beads and Bioplex 5.0 software. The detection antibody mix was optimized for human cell culture samples. Limit of detection (LOD) was measured using the following method: the standard deviation of the blank was multiplied by 2, and that value was added to the mean fluorescence intensity (MFI) of the blank value. The resulting value was then compared to the MFI values of the standards using the last 3 points. Based on the curve of the 3 standard points, the LOD is calculated using the MFI value plus two standard deviations.

IHC on formalin-fixed, paraffin embedded sections was performed as follows after deparaffinization as described (Tomassetti et al., 2009). To block endogenous peroxidase, sections were incubated with 3% hydrogen peroxide solution for 10 min at room temperature. For EGFR staining, antigen retrieval was carried out in 1 mM EDTA buffer (pH 8) for 10 min at 95°C in a pressure-cooker and then left at room temperature for 30 min. Samples were incubated in blocking solution (5% BSA/TBS) for 40 min at room temperature. The primary rabbit polyclonal anti-human

EGFR antibody was diluted 1:150. For IL-6 staining, antigen retrieval was carried out in 10 mM citrate buffer (pH 6) for 10 min at 95°C in a pressure-cooker, and then left at room temperature for 30 min. Blocking solution was 10% BSA/TBS, 0.025% Triton X-100 for 1 hr at RT. The primary rabbit polyclonal anti-human IL-6 antibody was diluted 1:400. For PAI-1 staining, antigen retrieval was carried out in 10 mM citrate buffer (pH 6) for 3 min at 120°C in a pressure-cooker, and then left at room temperature for 30 min. Blocking was carried out in 10% BSA/TBS, 0.1% Triton X-

100 for 30 min at room temperature. The primary rabbit polyclonal anti-human PAI-1 antibody was diluted to 3 g/ml. Incubation with each of the primary antibodies was performed overnight at 4ºC, slides were then incubated for 30 min at room temperature with the secondary biotinylated antibodies diluted 1:200. Slides were washed with PBS and peroxidase activity was revealed by incubating sections in DAB (3-3′diaminobenzidine) (DAKO, Denmark) for 5 min. After washing with water, sections were counterstained with Gill’s hematoxylin solution for 5 sec.

In silico analysis of EOC datasets

Before analysis, samples of dataset I (GSE9899) were filtered for "malignant", primary site "ovary" and histological type "serous", and the remaining 204 samples were used. Samples of dataset II

(GSE12172) (Anglesio et al., 2008), already normalized by RMA, were filtered for type

“malignant”. The filtered dataset was composed of 60 samples. Dataset III (GSE3149) (Bild et al.,

2006) was normalized using the RMA algorithm. Outlier and anomalous samples were filtered out, and the remaining 132 samples were used for analyses. For dataset IV, the raw data were downloaded from http://data.genome.duke.edu/earlystageovc (Berchuck et al., 2009), borderline and anomalous samples were filtered out and the remaining 78 samples were used. The data from datasets I and II were produced using the Affymetrix HG-U133 Plus 2 arrays, and those from datasets III and IV with the Affymetrix HG-U133A arrays.

LEGENDS TO SUPPLEMENTARY FIGURES

Fig. 1. EGFR membrane staining was determined by flow cytometry on EOC cell lines. The gray and black peaks represent the fluorescence of the cells alone or incubated with an isotype matched unrelated antibody. The purple peaks represent the fluorescence of cells incubated with anti-EGFR antibodies. The numbers above the histograms represent the percentage of mean fluorescence intensity. B. IL-6 release was assayed by ELISA in media for 24 hr from EOC cells grown for 24 hr in medium supplemented with 10% FCS. C. Western blot analysis was performed on total cell lysates from IGROV1 and OAW42 cells. After 24 hr of serum starvation, cells were left untreated or treated with 20 ng/ml EGF alone from 5 min to 60 min. The antibodies used are indicated. β- actin is shown as a control for protein loading. A representative experiment of 3 is shown.

Fig. 2. A. Gene expression intensities of EGFR, IL-6, PAI-1, and IL-8 on dataset I for each of the

204 cases; EGFR expression is reported on the right Y axis, and the others on the left Y axis. B.

Correlation between IL-6 and PAI- was analyzed in EOC datasets II, III, and IV. The values are plotted as log2 scale. Pearson correlations (r), linear regression and P values are reported.

Fig. 3. A. Distribution of IL-6 and PAI-1 levels in the EOC ascites containing tumor cells alone

(open circle), together with immune cells (filled circle) or containing only immune cells (filled square). REFERENCES TO SUPPLEMENTARY MATERIALS AND METHODS

Anglesio MS, Arnold JM, George J, Tinker AV, Tothill R, Waddell N et al. Mutation of ERBB2 provides a novel alternative mechanism for the ubiquitous activation of RAS-MAPK in ovarian serous low malignant potential tumors. Mol Cancer Res 2008; 6:1678-90.

Berchuck A, Iversen ES, Luo J, Clarke JP, Horne H, Levine DA et al. Microarray analysis of early stage serous ovarian cancers shows profiles predictive of favorable outcome. Clin Cancer Res 2009;

15:2448-55.

Bild AH, Yao G, Chang JT, Wang Q, Potti A, Chasse D et al. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 2006; 439:353-7.

Tomassetti A, De Santis G, Castellano G, Miotti S, Mazzi M, Tomasoni D et al. Variant HNF1

Modulates Epithelial Plasticity of Normal and Transformed Ovary Cells. Neoplasia 2008; 10:1481-

92. Supplementary Table 1. Characteristics of the EOC patients evaluated in the present study.

Presence of: Sample Histotype Grading FIGOa Tumor Immune Mesothelial ID Stage cellsb cellsb cellsb 1 Serous G3 IV Abundant Absent Present 2 Serous G3 IIIC Abundant Present Present 3 Serous G3 IIIC Abundant Rare Present 4 Serous G3 III Rare Abundant Present 5 Serous G3 IIIC Abundant Abundant Abundant 6 Serous G3 IIIC Present Present Present 7 Serous G3 IIIC Rare Present Abundant Serous and 8 G3 IV Abundant Present Present endometroid 9 Serous G3 IIIC Abundant Present Rare Mullerian 10 NA IIIC Present Rare Abundant mixed 11 Serous G3 IIIC Rare Present Present 12 Serous G2 IIIC Abundant Absent Present 13 Serous G2 IIIC Abundant Present NA 14 Serous G3 IIIC Rare Abundant Abundant 15 Endometroid G3 IV Rare Absent Abundant 16 Serous G3 IIIC/IV Present Abundant Abundant 17 Serous G3 IV Abundant Present Rare 18 Serous G3 IIIC Abundant Absent Absent 19 Endometroid NA IIIC Present Absent Abundant 20 Serous NA IIIC Abundant Abundant Rare 21 Serous G3 IIIC Present Absent Present 22 Serous G2/G3 IIIC Abundant Absent Rare 23 Serous G3 IIIC Abundant Present Present a Federation of Gynecologists and Obstetricians. bThe amount of cells present in ascites of EOC patients as defined by the cytopathologist at diagnosis. Supplementary Table 2. Measuraments of cyto/chemokines by Bioplex technology.

CYTO/CHEMOKINE1

TNF- IL- MIP- FGF- IL12 TGF- TNF- GRO- IL- α IL-1β IL-5 IL-7 12p70 IFN-γ G-CSF 1β MCP-1 basic IL-4 p40 β β MIP-1α MCP-3 IL-1α α IL-1RA 17F MIP-3α VEGF

Starved 4 hr *1.59 *0.45 und *0.37 *0.30 *2.12 2.96 *0.18 *1.57 47.53 *0.86 *0.70 *0.46 2.62 5.64 *1.10 *1.65 *1.43 160.24 *0.10 *0.17 85.2

EGF 4 hr *1.89 *0.42 *0.22 *0.39 *0.28 *1.57 3.17 und *2.04 45.46 *0.86 *0.72 *0.47 2.53 *2.13 *0.73 *1.76 und 155.45 und *0.01 114.78

EGF/AG1478 4 hr *1.51 *0.50 und *0.34 *0.29 *1.65 2.65 *0.48 *1.89 46.21 *0.97 *0.73 *0.63 2.58 3.89 *0.53 *1.73 *1.14 155.45 und *0.27 68.57

AG1478 4 hr *1.36 *0.43 und *0.37 *0.28 *2.03 *1.30 *0.11 *0.96 48.83 *1.01 *0.68 *0.53 2.79 6.66 *0.60 *1.84 *1.68 159.05 und *0.15 75.07

Starved 8 hr *1.55 *0.47 *0.50 *0.37 *0.27 *1.86 2.44 *0.16 2.49 52.31 *0.97 *0.71 *0.27 2.66 6.4 *0.80 *2.42 2.12 169.76 und *0.02 129.99

EGF 8 hr *1.93 *0.43 *0.36 *0.38 *0.30 *0.71 3.17 und *2.04 43.74 *0.93 *0.66 *0.26 2.45 *0.65 *1.13 *2.11 2.32 182.73 und *0.10 154

EGF/AG1478 8 hr *1.49 *0.47 und *0.34 *0.25 *2.38 2.34 und *1.57 47.9 *1.01 *0.69 *0.71 2.58 6.75 *0.73 *2.11 *1.68 168.57 *0.10 *0.29 69.68

AG1478 8 hr *1.74 *0.49 und *0.39 *0.30 2.64 *1.82 *0.50 2.92 52.13 *1.01 *0.64 *0.79 2.55 6.58 *0.80 *2.24 *1.68 173.9 *0.26 *0.21 81.42

Starved 24 hr *1.15 *0.39 *0.92 *0.47 *0.27 *0.40 *1.09 *0.21 *1.73 48.64 *1.01 *0.61 *0.16 2.41 und *1.59 3.04 *1.43 181.55 und *0.05 300.14

EGF 24 hr *1.59 *0.35 *1.28 *0.44 *0.27 *0.07 *0.89 und *1.40 43.93 *0.97 *0.58 *0.00 *2.22 und *1.39 3.41 2.32 162.63 und und 340.59

EGF/AG1478 24 hr *1.59 *0.52 *0.22 *0.37 *0.30 *2.29 *1.30 *0.60 2.64 51.4 *1.11 *0.66 *0.55 2.6 5.84 *0.87 *2.32 *1.14 176.85 *0.03 *0.47 115.13

AG1478 24 hr *1.62 *0.51 und *0.44 *0.29 2.46 3.38 *0.36 *2.19 50.49 *1.01 *0.60 *0.78 2.77 8.24 *0.67 *2.26 *0.30 172.13 *0.42 *0.45 122.64

Medium1 *2.34 *0.62 *0.50 *0.39 *0.34 4.28 7.17 *1.12 *1.89 53.4 *1.21 *0.70 *0.61 3.19 12.14 *0.60 *1.00 *0.99 163.22 *0.76 2.67 *1.17 CYTO/CHEMOKINE1 sCD40 I- RANTES IFN-α HGF EGF PAI-1 SAA Ligand TAC MIF TGF-α M-CSF SDF-1a IL-2 IL-6 IL-8 IL-10 IL-13 MMP-9 MMP-7 MMP-2 MMP-3 Starved 4 hr *0.44 6.14 *0.34 *1.19 143.37 13.45 *6.97 6.6 935.22 *0.61 106.12 27.99 *0.37 109.82 8.32 *0.52 *0.36 63.88 10832 23.57 17.61 EGF 4 hr *0.48 6.92 *0.38 911.4 189.84 20.07 *5.75 8.47 1062.64 *0.81 131.2 31.67 *0.37 199 15.57 *0.58 *0.28 109.95 12633 34.34 20.9 EGF/AG1478 4 hr *0.48 6.27 *0.23 962.55 148.02 19.51 *6.88 7.43 707.84 *0.74 108.54 29.5 *0.37 90.38 9.19 *0.58 *0.26 142.67 12591 51.82 21.42 AG1478 4 hr *0.43 6.14 *0.23 *1.37 150.38 17.11 *5.99 7.25 712.76 *0.74 113.39 29.71 *0.37 100.7 10.24 *0.50 *0.30 39.59 9974.1 12.94 14.77 Starved 8 hr *0.48 6.14 *0.47 *1.45 199.8 19.32 *6.27 11.6 816.38 *0.66 128.1 32.76 *0.35 191.18 13.39 *0.50 *0.34 11.94 10824 *1.68 14.77 EGF 8 hr *0.47 4.84 *0.43 869.23 296.84 19.51 *5.56 11.73 1326.5 *0.84 134.3 37.17 *0.42 306.74 21.8 *0.50 *0.19 82.39 15108 30.66 20.86 EGF/AG1478 8 hr *0.46 5.62 *0.25 942.7 144.46 19.51 *6.72 5.12 1001.7 *0.63 103.71 27.88 *0.38 86.37 9.19 *0.56 *0.30 87.05 9736.1 27.07 16.53 AG1478 8 hr *0.49 7.05 *0.32 *1.33 144.23 16.93 *7.89 6.95 902.81 *0.69 106.12 29.06 *0.38 104.44 11.73 *0.61 *0.41 180.11 12092 41.93 23.72 Starved 24 hr *0.54 6.14 *0.47 *1.03 366.51 20.26 *6.07 25.28 5586.42 *0.67 233.17 41.97 *0.34 376 26.65 *0.49 *0.34 49.03 12733 9.96 19.82 EGF 24 hr *0.44 4.06 *0.49 595.12 1225.76 18.57 *3.84 31.9 7183.85 *1.28 393.84 50.02 *0.31 500.4 54.94 *0.48 *0.26 15.96 13452 10.69 21.94 EGF/AG1478 24 hr *0.48 5.62 *0.23 1672.79 144.97 18.02 *6.80 6.69 3251.92 *0.84 136.79 30.91 *0.38 97.43 9.72 *0.71 *0.34 73.29 9963.8 20.17 16.28 AG1478 24 hr *0.49 5.62 *0.26 *2.19 154.1 15.33 *7.98 7.69 3968.68 *0.66 131.82 31.02 *0.38 100.47 11.29 *0.58 *0.34 127.51 11572 28.85 16.02 Medium1 *0.51 6.92 *0.26 *1.50 und 13.79 10.38 *0.49 9.17 *0.77 82.38 25.2 *0.40 *0.54 2.84 *0.65 *0.52 71.38 *9.58 12.94 und *Value interpolated beyond standard range 1Concentration are expressed in pg/ml Supplementary Table 3. Characteristics of EOC gene expression datasets used.

Dataset Totala Ib IIb IIIb IVb NAc

I 204 9 9 168 17 0

II 60 2 2 47 9 0

III 132 3 4 103 20 2

IV 41 0 0 31 10 0 a Number of patients b FIGO stage c Not available