Fluorescence imaging proteometry of thymosin β4 in single cells for detection of bladder transitional cell carcinoma

Abstract

4745 Background: Thymosin β4 (Tβ4), a ubiquitous 5 kDa peptide, functions both as an intracellular actin remodeling peptide that binds G-actin and an extracellular cytokine-like molecule that induces metalloproteinases and angiogenesis. Human transcriptome analyses have demonstrated increased expression of Tβ4 in renal-cell carcinoma, breast cancer, metastatic melanoma, and non-metastatic colon cancer. In this study, we developed fluorescence imaging proteometry (FIP) for analysis of single-cell Tβ4 as a potential biomarker of bladder transitional cell carcinoma (BTCC). Methods: Cancer cell lines and cells in urine and bladder-wash specimens were fixed and stored at -80o C. Specimens imprinted to slides were stoichiometrically labeled with anti-Tβ4 antibody (1o Ab) and fluorophore-tagged 2o Ab (Fl-Ab) or with 1o Ab, biotinylated 2o Ab and fluorophore-tagged streptavidin (Fl-SA), with a BioGenex i6000 Autostainer. Gray scale images were captured with an automated Leica microscope and a high-resolution CCD b/w camera that were benchmarked with standard fluorescent beads, before each image capture session. Fluorescence emission was quantified as mean fluorescence intensity (MFI) per cell, using Image-Pro Plus software. Average MFI (AMFI) was determined with 500-1000 cells. Results: Reliable Tβ4 FIP was achieved with optimized specimen fixation, storage, imprinting, labeling, and signal stabilization; inter-analysis normalization standards; and image analysis benchmarking. Thus, fixation with 0.5% formaldehyde, zinc-based S.T.F. Streck fixative, and 4% paraformaldehyde produced relative AMFIs of 100, 36, and 46, respectively. AMFI doubled with specimen exposure to 1o Ab for 15–20 hours at 4o C vs. 1 hour at 25o C, followed by Fl-Ab for 1 hour at 25o C. Tβ4 FIP provided intra- and inter-analysis reproducibility of ± 10% and routine resolution of a 20% difference in single-cell Tβ4. In an initial test, Tβ4 signal was present in normal bladder cells but absent from high-grade tumor cells. Conclusions: Sensitive and reproducible FIP has been established for analyzing Tβ4 expression in individual bladder cells, as a potential biomarker that may be decreased in BTCC. No significant financial relationships to disclose.