Time Gated Luminescence Imaging of Immunolabeled Human Tissues.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (∼nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Förster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.

In Situ Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay.

Protein function is often regulated by protein-protein interactions and post-translational modifications. Detection of these important biological phenomena in fixed biological samples could serve as an invaluable tool in biomedical research, drug development, as well as clinical cancer diagnostics and prognostics. We report here a novel methodology which utilizes unique antibody bioconjugates capable of forming proximity induced chemical ligation to enable in situ detection of proximal targets in fixed biological samples. Using this new methodology, we demonstrate in situ visualization of various protein heterodimers/complexes and post-translational modifications such as phosphorylation and ubiquitination. This new method offers high specificity, sensitivity, flexibility, and ease of use. In addition, the assay preserves critical contextual and heterogeneity information on biomarkers in clinically relevant samples.

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates.

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.

Enzymatically amplified mass tags for tissue mass spectrometry imaging.

Tissue mass spectrometry imaging (MSI) is a rapidly developing technology which promises to provide biomarker molecular information within tissue context, which is an unmet medical need in the era of personalized medicine. However, challenges associated with tissue specimens as well as the MSI technical limitations have hindered the practical applications of this technology. We report here a mass tag based MSI method that combines the strength of signal amplification by immuno-enzymatic reactions with the superior detection characteristics of mass spectrometry to enable matrix-free MSI of protein biomarkers in formalin fixed paraffin embedded (FFPE) tissues. The method involves binding of the target protein with a primary antibody with high affinity and specificity, followed by binding with a secondary antibody-enzyme conjugate. Enzyme substrates suitable for mass spectrometry detection are locally deposited at the site of the target through enzymatically catalyzed transformation. The precipitates thus serve as mass tags to be detected in mass spectrometry to represent the target biomolecule in tissue. Two enzymes and various substrates have been identified and successfully used to demonstrate the feasibility of this novel MSI method to image protein targets in FFPE tissue samples.

Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models.

Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.

Microwave-mediated synthesis of labeled nucleotides with utility in the synthesis of DNA probes.

A novel method of linking haptens to deoxycytidine 5'-triphosphate via microwave-mediated bisulfate-catalyzed transamination with hydrazine has been developed. This method enables the tethering of small molecule haptens to dCTP via a discrete polyethylene glycol (PEG) spacer, yielding N(4)-aminodeoxycytidine 5'-triphosphate-dPEG-haptens. This synthetic approach employs microwave-catalyzed hydrazinolysis that enables the attachment of spacers via hydrazine linkages. The microwave-mediated introduction of this hydrazine handle provides a significant improvement in yield over those of published thermal methods. The microwave reaction was shown to be scalable, and the final product was amenable to labeling with a wide variety of haptens. The resulting nucleotide triphosphates, N(4)-aminodeoxycytidine 5'-triphosphate-dPEG-haptens, can serve as unique substrates for the enzyme-mediated labeling of DNA probes. The efficacy of incorporation of one such novel nucleotide, N(4)-aminodeoxycytidine 5'-triphosphate-dPEG(4)-DNP, has been demonstrated in nick translation labeling of HER2 and HPV probes. The labeled probes have been shown to be effective in visualizing their target genes in tissue.

A technique for relative quantitation of cancer biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissue using stable-isotope-label based mass spectrometry imaging (SILMSI).

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin-fixed paraffin-embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix-free laser desorption ionization time-of-flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.