Protein array analysis

Angiogenesis related proteins were analysed using the Proteome Profiler Human Angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA), which allows simultaneous analysis of 55 proteins (soluble growth and differentiation factors, extracellular matrix components, proteases, membrane-bound receptors, and intracellular signalling molecules), shown in Table 1. The antibody array is a membrane based sandwich immunoassay using chemiluminescence for visualization according to standard immunoblotting procedures. The preparation and incubation of the arrays were performed as recommended by the manufacturer, and as previously reported [23]. To optimise the exposure time, allowing the detection and pixel quantification of a maximum number of protein spots, we initially tested six different exposure times, ranging from 30 s to 60 min, for each membrane. In the final analyses, we decided to use the 3 min and 30 min exposures (see Results). The developed array films were scanned, size adjusted, inverted in Adobe Photoshop, overlaid with a template and imported into Image J (https://imagej.nih.gov/lj) for quantification of the pixel intensity in each spot (S1 Fig). An average signal was calculated of the duplicate spots representing each protein, followed by background subtraction of a clear area of the membrane. Finally, each protein specific signal was divided by the average signal of the six positive reference spots of the membrane. The resulting normalized ratios, henceforward referred to as protein levels, were used in the subsequent analyses.

Proteins analysed by the Human angiogenesis antibody array (n=55), in alphabetical order. Serum samples collected at BL, baseline before treatment start; SOM, start of maintenance, after induction chemotherapy + bevacizumab before first cycle of bevacizumab maintenance and at EOT, End of treatment, after last cycle of bevacizumab at progression on maintenance therapy. ND, protein not detectable in any patient sample (0%).