Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology

Abstract

S U M M A R Y Detection and quantitation of phosphoproteins (PPs) in fixed tissues will become increasingly important as additional inhibitors of protein kinases enter clinical use and new disease entities are defined by molecular changes affecting PP levels. We characterize fixation conditions suitable for accurate PP quantitation that are achievable in a clinical laboratory and illustrate the utility of in situ quantitation of PPs by quantum dot (QD) nanocrystals in two models: (1) a therapeutic model demonstrating effects of a targeted therapeutic (quantitative reduction of phospho-GSK3b) in xenografts treated with enzastaurin; and (2) a diagnostic model that identifies elevated levels of nuclear phospho-STAT5 in routine bone marrow biopsies from patients with acute myeloid leukemia based on the presence of the activating FLT3-ITD mutation. Finally, we document production of a wellcharacterized tissue microarray of widely available cell lines as a multilevel calibrator for validating numerous phosphoprotein assays. QD immunofluorescence is an ideal method for in situ quantitation of PPs in fixed samples, providing valuable cell type-specific and subcellular information about pathway activation in primary tissues. (J Histochem Cytochem 57:701-708, 2009)

Phosphoproteins and the Dawn of Functional Phenotyping

“…Indeed, we observed that pSTAT5 in AML blasts with FLT3/ITD mutation as assessed by IHC in bone marrow biopsies is particularly strong compared to AML blasts with wild-type FLT3 [14] . We confirmed these qualitative data and using immunofluorescent assay showed a significantly higher nuclear expression of pSTAT5 in FLT3/ITD-positive cases compared to FLT3 wild-type cases [15] . In a similar line of thought, we studied another disease with constitutive tyrosine kinase activation, i.e.…”

“…Thus, fixed tissues must be used. Comparison of 5 commercially available fixatives showed that neutral buffered formalin fixation for durations typically used in clinical practice is satisfactory for quantitative assays [15] . Recently, a non-cross-linking (Streck; Omaha, Nebr., USA) tissue fixative was reported to significantly enhance the staining intensity of PPs compared with formalin [22] .…”

“…For PPs, the use of cell lines manipulated to produce high and low levels (even complete absence) of intended targets or cell lines known to be positive and negative for particular PPs may be useful to confirm specificity. Such information is available for many targets [15,26,27] , but more published data is needed (see Generation of Robust Control Tissue Samples below). If the antibody is suitable for WB, simultaneous WB (cell lysates) and IHC or IF (cytospins or cell blocks) analyses can be performed.…”

Advances in the application of nanotechnology in the diagnosis and treatment of gastrointestinal tumors

“…The detection limit was as low as 10 colorectal cancer cells per ml (31). Apart from circulating tumor cell detection, Bodo et al (32) demonstrated that QD immunofluorescence had the possibility of in situ quantitation of phosphoproteins in fixed samples, providing a promising method for highly cell type-specific detection application in the future.…”

Selection and Validation of Antibodies for Signal Transduction Immunohistochemistry

“…2b) a particular signal transduction protein may be useful to confirm the specificity and sensitivity. Such information is available for some phosphoproteins (12,15,16), but more published data are needed. Based on the same principles, validated antibody can be used for the truly quantitative analysis, using immunofluorescence assay (Fig.…”