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

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.

Mutual regulation of metabolic processes and proinflammatory NF-κB signaling

“…At the single-cell level, these various activation states are not phased in and can exist side by side, creating an even larger repertoire of fine-tuning and timing of NF-kB responses. 20 NF-kB activation in response to DNA damage proceeds by the atypical activation pathway, whereas immunologically relevant induction steps proceed via into the canonical and noncanonical NF-kB signaling pathways. 21 The canonical pathway is triggered by receptors sensing molecular patterns or by cytokine receptors, and the respective signal transduction proceeds by the regulated assembly of adapter proteins and enzymes mediating posttranslational modifications including ubiquitination and phosphorylation.…”

Regulation of Transcription Factor NF-κB in Its Natural Habitat: The Nucleus

“…One reason is that most data have been obtained from large cell populations characterized by heterogeneous NF-κB activation patterns [ 95 ]. Comparative analysis of single-cell epigenomic landscapes, NF-κB activation, and transcriptional responses will thus enable determining the precise temporal order of regulatory events and allow establishing causal relations between the diverse regulatory steps [ 96 , 97 , 98 ]. Causal dependencies between NF-κB and chromatin functions will become better addressable by advanced loss-of-function approaches allowing the rapid (and reversible) inactivation of signaling proteins using conditional degrons [ 99 ] or causing the rapid relocalization of proteins using anchor-away systems or related techniques [ 100 ].…”

Monitoring the Levels of Cellular NF-κB Activation States

“…Complementary detection oligos coupled to fluorochromes hybridize to repeating sequences in the amplicons and the fluorescent dyes can be detected by fluorescence microscopy and thus indicate the occurrence and intracellular localization of protein/protein interactions [ 120 ]. We used this methodology to detect p65/IκBα complexes mainly in the cytosol of cells, as revealed by fluorescence microscopy and its quantitative and statistical analysis [ 121 ]. In addition, dynamic changes were observed, as administration of IL-1α resulted in a significant decrease of p65/IκBα complexes, followed by the re-formation of these complexes 90 min after the addition of the stimulus [ 121 ].…”

“…We used this methodology to detect p65/IκBα complexes mainly in the cytosol of cells, as revealed by fluorescence microscopy and its quantitative and statistical analysis [ 121 ]. In addition, dynamic changes were observed, as administration of IL-1α resulted in a significant decrease of p65/IκBα complexes, followed by the re-formation of these complexes 90 min after the addition of the stimulus [ 121 ]. This experimental approach also allowed to visualize p50/p65 dimers in the nucleus and can be used to detect any other interesting protein/protein complex and also post-translational modifications, provided that suitable antibodies from two different species are available.…”