Abstract
The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.
Highlights
Epithelial ovarian cancer (EOC) is the fifth-leading cause of cancer-related deaths in women, with a higher fatality-to-case ratio than any other gynecologic malignancy in the United States. [1,2,3] A major problem is that greater than two-thirds of EOC cases are diagnosed at advanced stages (Stages 3 or 4), when fiveyear survival is about 33%
Xenograft mouse plasma pooled from four mice with the largest tumors was subjected to extensive fractionation (180 fractions) using a 4 D plasma proteome separation method developed in our laboratory which consists of immunoaffinity depletion of major serum proteins, MicroSol IEF, 1D Sodium dodecyl sulfate (SDS)-PAGE, and LC-MS/MS
The presence of human proteins in the plasma demonstrated these proteins were produced by the tumor and shed into the blood, but many of these assignments were based upon only a few peptides
Summary
Epithelial ovarian cancer (EOC) is the fifth-leading cause of cancer-related deaths in women, with a higher fatality-to-case ratio than any other gynecologic malignancy in the United States. [1,2,3] A major problem is that greater than two-thirds of EOC cases are diagnosed at advanced stages (Stages 3 or 4), when fiveyear survival is about 33%. Comparison of patient and control serum or plasma to discover biomarkers is complicated by the fact that cancers and other conditions induce inflammatory responses involving changes in abundance of multiple blood proteins, and these changes are not very specific to a single disease. [10] Identifying cancer-specific changes in the context of this great complexity and inflammation-induced variability is very difficult when patient serum samples are directly analyzed to discover new biomarkers using proteomics For this reason, many investigators have turned to alternative strategies for initial discovery of candidate biomarkers, including proteome analysis of: EOC cell lines, cell surface proteins in EOC cell lines, proteins shed by these cells into the media (the secretome), and patient ascites. Changes in abundance levels of a protein in the tumor tissue do not necessarily correlate with their abundance levels in the blood
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