Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays.

Mayr-Buro Mayr-Buro,Schlereth Schlereth,Meier-Soelch Meier-Soelch,Beuerlein Beuerlein,Schmitz Schmitz,Tenekeci Tenekeci,Kracht Kracht

doi:10.3390/cancers11081199

Abstract

The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine.

Highlights

Advanced cancer is characterized by an increasing degree of tumor cell heterogeneity, resulting in tumor cell populations that display distinct signaling pathways and molecular signatures [1].This non-uniform distribution ofgenetically different tumor-cell subpopulations appears as both temporal and spatial heterogeneity, in the process of disease progression or within disease sites, respectively [2]
As an important additional control for the specificity of proximity ligation assays (PLAs), these experiments were performed in p65 knockout
The number of PLA signals was strongly diminished in the p65 knockout cells or after staining of the mouse embryonic fibroblasts (MEFs) only with single antibodies or omitting both primary antibodies, as revealed by fluorescence microscopy (Figure 1B) and its quantitative and statistical analysis (Figure 1C,D)

Summary

Introduction

Advanced cancer is characterized by an increasing degree of tumor cell heterogeneity, resulting in tumor cell populations that display distinct signaling pathways and molecular signatures [1]. This non-uniform distribution of (epi)genetically different tumor-cell subpopulations appears as both temporal and spatial heterogeneity, in the process of disease progression or within disease sites, respectively [2]. Genetic instability is one of the hallmarks of cancer, allowing the rapid evolution of subclones [3]. NF-κB signaling is often suppressed in tumor-associated immune cells such as TAMs and cytotoxic T-cells, which compromises efficient antitumor immunity [9]

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Discussion

Conclusion