Although still very much under development, the antibody microarray technique has already shown wide application potential for clinical cancer research and diagnostics [45
]. Table lists some recent applications of antibody microarrays in oncoproteomics. The antibody platforms had either been fabricated in-house or obtained from commercial sources. The number of binders varied from a few tens, as in the analysis of cytokine networks [46
] or functional pathways [47
], to hundreds, as in studies focused on a more global protein expression analysis [29
]. Several sources of samples have been used, including culture cell extracts [29
], dissected tissue biopsies [25
], exhaled breath [46
] and body fluids [30
]. Nevertheless, the most studied specimens were sera taken from both cancer patients and healthy controls [30
]. The rationale is that serum reflects the body's whole cellular metabolic harvest, and leakage of proteins from a particular organ or group of cells to the circulation provides some reflection of biochemical alterations during disease. In addition, in more technical terms, protein complexity is relatively low in serum and protein extraction is easy to perform.
Application of antibody microarrays in cancer research
Hudelist et al.
] used antibody microarrays for profiling expressed proteins in normal and malignant breast tissues. They found increased expression levels of several proteins in malignant breast tissues, such as casein kinase Ie, p53, annexin XI, the cell-cycle protein CDC25C, the general transcription initiation factor eIF-4E and mitogen-activated protein (MAP) kinase 7, using commercial arrays of 378 antibodies. In another report [61
], 224 antibodies revealed proteins that are related to doxorubicin therapy resistance in breast cancer cell lines. A decrease in the expression of MAP kinase-activated monophosphotyrosine, cyclin D2, cytokeratin 18, cyclin B1 and heterogeneous nuclear ribonucleoprotein m3-m4 was found to be associated with doxorubicin resistance. Other recent investigations helped identify a marker involved in invasion (interleukin (IL)-8) [62
]. Studying the serum proteome from metastatic breast cancer patients and healthy controls with recombinant single-chain variable fragment (scFv) microarrays [54
], breast cancer was identified with a specificity and sensitivity of 85% on the basis of 129 serum analytes.
In bladder cancer, an array of 254 antibodies showed 93.7% sensitivity to discriminate between serum samples of 58 healthy subjects versus 37 bladder cancer patients [53
]. The impact of radiation treatment was evaluated in LoVo colon carcinoma cells [63
]. An array of 146 antibodies showed increased expression of apoptosis regulators paralleled by downregulation of CEA, pointing to a possible application for monitoring response to radiation therapy in colon cancer. In colorectal cancer, the marker IPO-38 [30
], cytokeratin 13, calcineurin, the serine/threonine kinase CHK1, clathrin light chain, MAP kinase 3, phosphoprotein tyrosine kinase 2 (also called focal adhesion kinase, phosphorylated at Ser-910) and the p53 regulator MDM2 [64
] were found as possible biomarkers. They were further validated with standard protocols such as ELISA, immunoblotting, immunohistochemistry and MALDI-TOF/TOF mass spectroscopy. However, the number of patients evaluated in these colorectal cancer studies was low. The application of antibody microarrays to prostate cancer also identified several potential marker proteins [65
]. Analysis of cytokines from prostate fluid of patients with minimal and maximal cancer volume revealed a possibility for early detection of the disease [67
Several publications have recently reported the use of antibody microarrays in assessing markers of lung cancer, which is the leading cancer-related cause of death. Kullmann et al.
] tested cytokine profiles with a 120-antibody array in breath condensates of 50 smoking lung cancer patients and 25 smokers without clinical or radiological sign of a pulmonary tumor and were able to differentiate the two groups by nine cytokines, including eotaxin, fibroblast growth factors, IL-10 and macrophage inflammatory protein (MIP)-3. However, the results were not stratified according to stages and histological subtypes owing to the use of pooled samples. Gao et al.
] constructed an array of 48 antibodies against distinctive serum proteins. They analyzed 24 newly diagnosed subjects with lung cancer, 24 healthy controls and 32 subjects with chronic obstructive pulmonary disease. C-reactive protein, serum amyloid A, mucin 1 and α1-antitrypsin were among the proteins that showed higher abundances in the lung cancer samples than in the control samples.
Pancreatic cancer has received much attention, being one of the most deadly forms of cancer with basically no current treatment available. Initial observations of serum profiles came from Haab and colleagues [59
], revealing individual and combined protein markers associated with pancreatic cancer and variations in specific glycans on multiple proteins. In another study from the same group [68
], antibody microarrays were used to analyze post-translation modification of serum protein in pancreatic cancer patients. By profiling both protein and glycan variations [69
], they found cancer-associated glycan alteration on the proteins MUC1 and CEA [68
]. The Borrebaeck group [56
] used an array of recombinant scFv antibodies in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus samples from healthy subjects. They reported a protein signature based on 19 non-redundant analytes discriminating between cancer patients and healthy subjects.