Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning

Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n = 22) taken at 6 weeks post-transplantation. Patients were stratified for IFTA development (<10% versus ≥10%) from 6 weeks to 6 months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios, and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at 6 weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development 6 months post-transplantation (p < 0.05). CD3+CD8−/CD3+CD8+ ratios were higher in patients with <10% IFTA development (p < 0.05). We observed a high correlation between CD163+ and CD4+GATA3+ cell density (R = 0.74, p < 0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi spectrally imaged slides of kidney transplant biopsies.

Multiplex Immunofluorescence Tyramide Signal Amplification for Immune Cell Profiling of Paraffin-Embedded Tumor Tissues

Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Tyramide Signal Amplification coupled with multiple immunolabeling and RNAscope in situ hybridization in formaldehyde-fixed paraffin-embedded human fetal brain.

Several strategies have been recently introduced to improve the practicality of multiple immunolabelling and RNA in situ hybridization methods. We present a modified hybrid protocol of recently described complex detection strategies: (1) elution of antibodies prior to second round of staining (2) use of integrated polymers of HRP with secondary antibodies, and (3) tyramide signal amplification of multiple immunofluorescence labelling, to achieve a high sensitivity sequential multiple labeling using antibodies from the same species. A modified protocol of the novel RNAscope in situ hybridization method, including coupling with immunofluorescence on sections of early human fetal brain, has also been developed. These two techniques, when properly optimized, were highly compatible with routine formaldehyde-fixed paraffin-embedded tissue that preserves the best morphological characteristics of delicate fetal brain samples, allowing high power signal amplification for detection of protein and mRNA of genes that are sparsely expressed in the human fetal telencephalon.

Dynamic nuclear lamina-chromatin interactions during G1 progression

A large fraction of heterochromatin in the metazoan genome is associated with the nuclear lamina (NL) in interphase nuclei. This heterochromatin is often referred to as Lamina-Associated Domains (LADs) and are often mapped from cell populations asynchronously progressing through the cell cycle. We and others have recently reported that LADs are largely stable during G1, S, or G2 phases of the cell cycle, and appear similar to LADs mapped from bulk cell populations. LADs in senescent cells, however, are reported to be quite different from proliferating cells, and it remains unclear how senescent cell LADs are established. As cells finish mitosis and re-enter G1, reassembly of the nuclear envelope and NL appears to precede mitotic chromosome decondensation. Therefore, the initial NL interactions with the decondensing chromatin may be quite different from those reported in asynchronous or FACS isolated G1, S, or G2 populations. By developing a modified version of the Tyramide-Signal Amplification sequencing (TSA-seq), which we call chromatin pull down-based Tyramide Signal Amplification-sequencing (cTSA-seq), we uncover a dynamic NL-chromatin interaction as cells progress through G1. The appearance of stable LADs coincides with sufficient chromatin decondensation and active gene expression in G1. Interestingly, early G1 NL-chromatin interactions, which are found toward the telomeric ends of human chromosomes, are similar to those found in oncogene-induced senescent cells. We find that the assembly of LADs during the formation of the G1 nucleus is gradual and that the arrest of NL-chromatin interactions in early G1 may contribute to genome disorganization of senescence cells.

Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.