The PD-1/PD-L1 signaling pathway is essential for immune control and maintaining immune system balance. PD-1 and PD-L1 proteins exert most functions in cells and tissues by undergoing modifications and forming dynamic complexes – effects that cannot be explored by genomics, transcriptomics, or conventional immunostaining methods. Numerous cancer therapies are being developed that affect PD-1/PD-L1 signaling, and tools to study the PD-1/PD-L1 axis are, therefore, essential. One prerequisite for successfully establishing diagnostic assays is reproducibility, standardization, and, ideally, high sample throughput via automation. Here, we aimed to introduce a new technological solution to allow for the simultaneous detection of PD-L1 and PD-1 interaction and the adjacent immune cell context in tissue samples on the Lunaphore’s automated microfluidic staining platform. The assay for detecting PD-1-PD-L1 interaction was developed and technically validated by Navinci Diagnostics. Although the detection of interaction alone provided important information on the immune status in the tumor microenvironment, we believed that PD-1/PD-L1 readout would be most powerful in the context of its cellular ecosystem. Therefore, the PD-1/PD-L1 assay was combined with the analysis of immune and cancer cells to determine their involvement in this signaling. The feasibility of combining multiplex immunofluorescence with proximity ligation assay was conducted and confirmed on human tonsils. The spatial profiling panel included immune markers for all T-cells (CD3), helper T-cell (CD4), cytotoxic T-cells (CD8), B-cells (CD19/20) and activation/exhaustion markers granzyme B (activated cytotoxic T-cell), Ki67 (proliferating cells), and LAG3 (malignant B cells, CD8 T cells, CD4 Tregs). We evaluated, implemented, and optimized the assays on the Lunaphore staining instrument to reduce labor time and cost for the end users. In the next phase, the potential of our assay to be benchmarked as a diagnostic tool in clinical praxis will be thoroughly tested and validated on diagnostic tissue samples from NSCLC patient cohorts. We believe this approach will enable spatial and functional studies of the interface between the tumor and the immune system and provide necessary information about signaling pathway activation in situ, the latest representing a novel state-of-the-art in tissue diagnostics. Citation Format: Hampus Elofsson, Agata Zieba Wicher, Carolina Oses Sepulveda, Tony Ullman, Maria-Giuseppina Procopio, Alix Faillétaz, Diego G. Dupouy, Charlotte Stadler. Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4336.
Determining the levels of protein-protein interactions (PPI) is essential for the understanding of signal transduction, regulation of gene expression and mutation effects, infection mechanisms, identification of diagnostic markers, etc. Taking the already challenging task of PPI identification a step further, the next aim is to determine the levels of non-interacting proteins in parallel. The ability to concurrently monitor free and interacting proteins opens the door to studying their functional states and interplay, thus gaining deeper insight from a single staining experiment. Additionally, to accomplish this in situ, retaining the structural integrity of the cell, helps understand spatiotemporal communication between proteins in their native environment. To this end, we designed Naveni TriFlex Cell – a highly sensitive and specific proximity-based technology, relying on two user-determined primary antibodies against the targets of interest, and on proprietary TriFlex Navenibodies, which are antibody-based proximity reagents. TriFlex Cell detects total protein A (i.e., both free and in complex with B), total protein B, and the AB interaction. The detected A, B and AB signals are amplified and generate fluorescent readout in three channels corresponding to each protein pool. As proof of principle, we stained MCF7 cells for E-cadherin and β-catenin, which are known to interact in the adherens junctions. We observed highly abundant interactions at the cell membrane, some complexes and free proteins in the cytoplasm, and very low background in controls where either or both primary antibodies were omitted. These results attested to the high sensitivity and specificity of our method. Next, we explored the expression and interaction of Histone H3 and Lamin B, a nuclear envelope protein. Despite the compact structure of the nucleus, we were able to distinguish individual signals and abundant interactions, with free Lamin B particularly enriched in the nuclear membrane. In dividing cells, we observed diffuse Lamin signals in line with nuclear envelope breakdown, and more densely packed Histone H3 associated with chromatin. In contrast to non-mitotic cells, interactions were few, but increasing with the advance from metaphase to telophase. Finally, we stained against GM130, a Golgi complex protein, and COX1, a mitochondrial marker, as a biological non-interaction control experiment. TriFlex Cell successfully detected the individual proteins in their respective subcellular locations, and no complexes. In summary, our data demonstrate that Naveni TriFlex Cell is a sensitive and specific method that visualizes low and high abundant targets in various cell compartments and adds value by uncovering protein interplay, such as complex formation/dissolution under dynamic biological conditions. Citation Format: Axel Klaesson, Doroteya Raykova, Agata Zieba Wicher. Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2021.
Immunofluorescent staining of tissues via in situ proximity ligation assay (isPLA) is now a well-established tool for highly sensitive detection of protein-protein interactions and their localization. It finds versatile applications in basic research, understanding of cellular processes, visualization of tissue architecture, and identification of biomarkers, among others. However, standard IF and isPLA techniques alike may suffer from high background. Tissues are particularly affected by background problems caused by autofluorescence and tissue infiltration of various cell types (e.g., immune, endothelial) where the fluorescently labelled oligos used for detection in isPLA may bind unspecifically. To address this, we developed a next generation isPLA-based technology for fluorescent detection of protein interactions in FFPE and fresh frozen human and mouse tissues, called NaveniFlex Tissue. In contrast to previously available protocols, it can visualize signal that would otherwise be obscured by background, thereby increasing sensitivity. Here, we compare the ability of NaveniFlex Tissue and similar commercial kits to detect the interaction between low abundance proteins Podocalyxin and Ezrin in the glomeruli of healthy kidney and in breast cancer, where co-expression is a prognostic marker for increased metastatic potential. While other kits generate high background which masks isPLA signal entirely, NaveniFlex Tissue dramatically reduces cell-based background and visualizes the interaction in its specific localization. Furthermore, in a TMA staining for the Mucin 16/Mesothelin interaction, our method successfully demonstrates strong and clear signal in stage III ovarian cancer. Moreover, it sensitively detects even low abundance interaction in a stage Ia tumor. In contrast, a different commercial kit detects significantly less interactions in the stage III tumor, while in the lower grade tumor it does not generate signals above background level. Therefore, NaveniFlex Tissue improves detection in tissues with varying disease progression, thus adding information and prognostic value to the staining. Taken together, our data illustrate that NaveniFlex Tissue outperforms current isPLA-based techniques by efficiently reducing background, which improves visualization of signal and signal-to-noise ratios in various healthy and diseased tissues. Citation Format: Hampus Elofsson, Doroteya Raykova, Agata Zieba Wicher. Powerful background reduction in fluorescent tissue stains with an improved proximity-based technology for detection of protein-protein interactions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6772.
Immune checkpoints (ICs), which are inhibitory signaling pathways that can down-modulate the immune responses of T cells, are pivotal in peripheral tissues and for maintaining immune self-tolerance. Among the many molecularly defined IC proteins, some of the most studied ones are CTLA-4 and its binding partners important for early T-cell co-inhibition, along with the PD-1/PD-L1 axis proteins that carry out late co-inhibitory signals. Notably, in addition to antigen presenting and other immune cells, many tumors also express CTLA-4 and PD-L1, which facilitate tumor evasion from the immune system. The inhibition of these IC proteins has revolutionized the field of cancer therapy, but its efficiency has been limited to a poorly defined subset of patients. Patient outcomes are likely to have a stronger correlation to high levels of PD-1/PD-L1 interaction and other hallmarks of pathway activation, such as PD-1 phosphorylation and recruitment of SHP-2, than to the overexpression of a single IC protein alone. To improve the predictive value of tissue staining, we created an immuno-oncology line of ultrasensitive kits based on proximity ligation, which detect the activation of multiple ICs. Naveni™ PD1/PD-L1 is a tool for direct visualization of the interaction between PD-1 and PD-L1, and has been verified in various FFPE tumor tissues. Naveni™ pY PD1 sensitively detects PD1 phosphorylation, which is the first step in the PD-1/PD-L1 inhibitory pathway activation. To study the subsequent recruitment of SHP-2 to the activated PD-1 receptor, which is crucial in immune cells but dispensable when the pathway is activated between two tumor cells, one can use the upcoming Naveni™ PD1/SHP-2 kit. Furthermore, we are developing assays to observe the competing interactions of CTLA-4 and CD28 with their binding partners CD80 and CD86, the interplay of which is pivotal for the initiation of early co-inhibition. All kits can be used on consecutive tissue sections to obtain a comprehensive picture of tumor IC pathway activation, which may not always correspond to CTLA-4, PD-1 or PD-L1 overexpression. While these kits have a chromogenic readout applicable to brightfield microscopy, we also have preliminary data on a PD-1/PD-L1 assay with a fluorescent readout thanks to which it can also be multiplexed. The multiplex feature generates an immune profile which may help improve immunotherapeutic strategies. The Naveni™ assays can be applied in basic research to elucidate the interplay of IC axes and downstream molecules, in pre-clinical and clinical research to compare stainings with the existing IHC assays and evaluate the potential prognostic value of interaction detection, and in pharma, aiding the development of new drugs or bispecific antibodies. Citation Format: Desirée Edén, Ka I Au Ieong, Naomi Cook, Doroteya Raykova, Agata Zieba Wicher. In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6372.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.