Hypothesis

Senescent cells do not merely broadcast inflammatory SASP; they create a spatially restricted “negotiator zone” in which adjacent cells undergo a specific interactome rewiring that enriches high‑betweenness, pathway‑bridging proteins. This rewiring equips neighbors with a transient, coordinated capacity to sense damage, activate autophagy, and limit proliferative spread, thereby functioning as an adaptive tissue‑scale checkpoint rather than a passive by‑stander.

Mechanistic rationale

SASP factors such as IL-1α and IL-8 trigger NF‑κB signaling in nearby cells 2, but proteomic studies show that NF‑κB also induces expression of scaffold proteins (e.g., paxillin, vinculin) and ubiquitin‑ligase adaptors that increase node betweenness in the protein‑protein interaction network. In C. elegans, genes with high betweenness centrality correlate with lifespan extension 3, suggesting that topologically central nodes are key regulators of stress resilience. Furthermore, pro‑longevity genes exhibit significantly higher PPI connectivity than anti‑longevity genes 1. We propose that SASP‑driven NF‑κB activity in neighbors selectively up‑regulates these scaffold/adaptor proteins, converting the local interactome into a hub‑rich state that integrates signals from DNA damage, oxidative stress, and growth factor pathways.

Testable predictions

1. Cells within ~2–3 cell diameters of a senescent focus will show significantly higher betweenness centrality for proteins linked to autophagy (e.g., ATG5, Beclin‑1) and DNA‑damage response (e.g., 53BP1, BRCA1) compared with cells farther away or in tissue lacking senescence.
2. Perturbing NF‑κB in the neighbor compartment (using a IKKβ inhibitor or conditional RelA knockout) will abolish the increase in betweenness centrality and the concomitant enrichment of autophagy/DNA‑repair modules without affecting SASP secretion from the senescent source.
3. Artificially boosting betweenness‑centrality proteins in neighbor cells (via inducible expression of a synthetic scaffold that links autophagy and ubiquitin‑ligase complexes) will phenocopy the protective effects of senescent cells, reducing hyperproliferation in a damage‑induced hyperplasia model even when senescent cells are cleared by senolytics.
4. In vivo spatial proteomics (BioID/APEX2 targeted to senescent cells) will reveal a radial gradient of biotinylated proteins where the peak of high‑betweenness nodes coincides with the zone of suppressed Ki‑67 staining and elevated LC3‑II conversion.

Experimental approach

• Model: Induce localized senescence in mouse ear epidermis using UV‑B irradiation or oncogenic RAS expression confined to a defined stripe; confirm senescence by SA‑β‑gal and p16^Ink4a^ staining.
• Proximity labeling: Express APEX2 fused to a senescence‑specific promoter (e.g., p16^Ink4a^-APEX2) to label proteins in senescent cells and their immediate extracellular milieu; alternatively, use a secreted biotin ligase (e.g., BirA*) fused to a SASP cytokine to capture neighbor‑proximal proteins.
• Mass spectrometry & network analysis: Quantify biotinylated proteins, construct PPI networks using BioGRID interactions 4, compute betweenness centrality for each node, and map enrichment of autophagy, DNA‑repair, and tissue‑remodeling modules via Gene Ontology.
• Perturbations: Treat cohorts with IKKβ inhibitor (e.g., BMS‑345541) or neighbor‑specific RelA floxed allele crossed to a Cre line activated by a paracrine reporter (e.g., p16‑LSL‑Cre). Assess changes in network topology and functional readouts (Ki‑67, cleaved caspase‑3, LC3‑II).
• Rescue: Express a designed scaffold (FKBP‑FRB dimerizable linker that binds ATG5 and TRAF6) in neighbor cells via a doxycycline‑inducible system; evaluate whether this restores the protective phenotype after senolytic clearance.

Potential outcomes and interpretation

• Supportive: Observation of a radially decaying gradient of high‑betweenness proteins that correlates with reduced proliferation and increased autophagy, which is lost upon NF‑κB blockade, would substantiate the negotiator‑zone model.
• Refutational: Uniform network topology across distances, or failure of NF‑κB inhibition to alter betweenness despite suppressed SASP signaling, would indicate that senescent cells act primarily through soluble factor toxicity rather than structuring a specialized interactome neighborhood.