Apelin, a novel predictive biomarker for bevacizumab response in colorectal cancer

Gene expression profiles of various isolated normal and tumour cell types The goal was to identify genes differentially expressed in different cell types between normal and tumour tissues. To this end, different cell types (endothelial cells, macrophages and epithelial cells) were isolated from non-paired primary normal colon tissues and colorectal carcinomas and subsequently RNA was isolated. Endothelial cells and epithelial cells were isolated using facs-sorting, whereas macrophages were isolated by means of plastic adherence. For comparison RNA was also isolated from non-paired whole normal colon tissue and whole tumour colorectal carcinomas (so called “bulk”). RNA was processed and hybridized onto Agilent microarrays. Primary goal was to establish an endothelial cell genetic profile. For this we compared the gene expression of tumour endothelial cells (TECs) with normal endothelial cells (NECs). The analysis consisted of 3 batches that were hybridized at 3 different time points. Besides colon tissues, but outside the scope of these analyses, also RNA of whole kidney or liver (metastasis) tissues and cell fractions were hybridized on these arrays. Some replicate samples within and between batches served as technical replicates and as reference within one batch. References for batch 2 were colon tumour bulk 94 and for batch 3 colon tumour bulk 121. Technical replicates were: colon adenocarcinoma 104 (n=2), colon tumour EC 109 (n=2), colon tumour EC 118 (n=2), colon tumour epithelial cells 124 (n=2), kidney normal EC 4 (n=2), kidney tumour EC 4 (n=2), kidney normal EC 5 (n=2), kidney normal EC 6 (n=2), colon normal EC 68 (n=2), kidney normal EC 7 (n=2), colon tumour EC 71 (n=2), liver metastasis 72 (n=2), colon tumour EC 80 (n=2), colon normal EC 81 (n=2), colon normal EC 82 (n=2), colon normal EC 89 (n=2), kidney tumour EC 8II (n=2), kidney normal EC 9 (n=2), colon tumour EC 94 (n=2), colon tumour bulk 99 (n=2), colon tumour EC 99 (n=2), colon tumour bulk 94 (n=3, reference batch 2), colon tumour bulk 121 (n=13, reference batch 3).