Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

Abstract

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.

Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs

“…The cells used for immunohistochemistry (IHC) were cultured in normal cell culture flasks according to protocols from respective distributors. The cells were harvested, and agarose cell gels were prepared for IHC (Andersson et al, 2006).…”

“…The cell microarrays were IHCstained in duplicate (Andersson et al, 2006), and the resulting images were annotated using an automated image analysis application (Strömberg et al, 2007). The staining patterns were divided into five groups (not representative, negative, weak, moderate, and strong expression) depending on the intensity of With this approach, staining patterns for nine msAbs were generated [MDR1, MRP1, MRP2, MRP4, ileal bile acid transporter (IBAT), PEPT1, MCT1, OCT2, and OATP1B3] (Uhlén et al, 2005).…”

Nuclear expression of the RNA-binding protein RBM3 is associated with an improved clinical outcome in breast cancer

“…org). 11,12 To date, more than 3000 antibodies (corresponding to more than 2600 different human proteins) have been screened in tissue microarrays, representing 48 types of normal tissues, 216 human tumor samples representing the 20 most common forms of human cancer and 47 cell lines, 13,14 In addition to generating a map of protein expression patterns, this atlas can also be used as a platform for the discovery of new diagnostic, prognostic and predictive cancer biomarkers. 12 One such candidate is RBM3, which, on the basis of its differential expression among the breast cancer samples in the protein atlas was further evaluated in tissue microarrays constructed from two independent breast cancer cohorts--one consecutive cohort (n ¼ 512) and one cohort containing tumors from a randomized tamoxifen trial conducted in premenopausal women (n ¼ 500).…”

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

“…The CMA construction required the initial preparation of cell paraffin blocks (Andersson et al 2006), which consisted of cell harvesting (about 1 × 10 6 cells) and incubation of the pellets for 20 min at 4C in 4% paraformaldehyde (PFA). The fixed cells were washed in PBS, resuspended in 100 µl of 2% low melting agarose, and finally applied in donut-shape agarose molds made up of 3% standard agarose.…”

“…• Predictive TMAs, used to test markers that predict drug response (Andersson et al 2006;Hewitt 2012) • Control TMAs, used to establish experimental protocols (Wan et al 1987 Depending on the number of samples to be analyzed, it is possible to choose among different instruments, ranging from a completely manual arrayer to a fully automated one. In a manual system (e.g., Beecher Manual Tissue Arrayer I [Beecher, Sun Prairie, WI], Tissue Arrayer MiniCore, Alphelys, France, and others), tissue cores are extruded from a selected area of the donor block and inserted directly in the TMA recipient block.…”