The mechanisms driving radioresistance in esophageal squamous cell carcinoma (ESCC), a major cause of treatment failure, are not fully elucidated. Based on our findings that link elevated acetylcholine (ACh) and its receptor subunit CHRNB4 to radioresistance and poor prognosis, this study aims to elucidate the molecular mechanisms by which ACh-mediated cholinergic signaling contributes to the radioresistant phenotype in ESCC.

        The mechanistic pathway was elucidated in radioresistant ESCC cell lines established through fractionated irradiation. The direct transcriptional regulation of ChAT by NF-κB was confirmed using chromatin immunoprecipitation-qPCR  and dual-luciferase reporter assays. The ChAT-ACh-Ca²⁺-NF-κB autocrine positive feedback loop was characterized using calcium imaging, immunofluorescence, and pharmacological inhibition. Functional consequences on migration, invasion, and stemness were evaluated by Transwell and sphere formation assays. Immune evasion was assessed by immunofluorescence and flow cytometry for surface CD47 expression, and through in vitro macrophage phagocytosis assays. Finally, a synthetic biology strategy was developed by engineering tumor-tropic E. coli Nissle 1917 to secrete an ACh-degrading enzyme (ChoE), and its synergistic radiosensitizing efficacy was evaluated in vitro.

        We first established that radiotherapy (RT) triggers robust NF-κB activation in ESCC cells, as evidenced by the marked degradation of its inhibitor, IκB, and a significant increase in the nuclear translocation of p65. Crucially, we linked this activation to the emergence of a pro-tumorigenic cholinergic phenotype. In our cell line models, RT led to a significant upregulation of choline acetyltransferase (ChAT) expression. To confirm a direct regulatory link, ChIP-qPCR and dual-luciferase reporter assays were performed, which demonstrated that following radiation, activated NF-κB directly binds to the ChAT promoter to drive its transcriptional activity.

Tumor-Derived ACh Establishes a Ca²⁺-Dependent Positive Feedback Loop that Sustains NF-κB Activity

        With elevated ChAT expression, ESCC cells began to synthesize and secrete ACh. We next demonstrated that this tumor-derived ACh establishes a self-reinforcing positive feedback loop by acting on α3β4 nicotinic receptors on the tumor cell surface. This engagement triggered a significant influx of intracellular Ca²⁺, which in turn activated downstream effectors, including CaMKII and calcineurin, ultimately promoting further nuclear translocation of NF-κB. Critically, pharmacological blockade of α3β4 receptors or chelation of intracellular Ca²⁺ effectively abrogated this feedback, leading to a significant reduction in NF-κB nuclear localization and activity.

Cholinergic Signaling Promotes EMT and Malignant Phenotypes through Crosstalk with the Wnt/β-catenin Pathway

        Beyond amplifying NF-κB, the sustained Ca²⁺ signal was found to crosstalk with the Wnt/β-catenin pathway. We observed enhanced β-catenin stabilization and nuclear translocation, leading to the subsequent upregulation of the EMT-driving transcription factors Snail and Slug. Functionally, this resulted in increased cellular migration, invasion, and the acquisition of cancer stem-like cell properties. Importantly, disruption of the cholinergic loop, either through α3β4 blockade or Ca²⁺ chelation, concomitantly attenuated this pro-metastatic axis, demonstrating its dependence on the ACh-driven signaling.

The NF-κB Axis Drives Immune Evasion via Transcriptional Upregulation of CD47

        We then investigated the role of this pathway in immune escape. Our results show that NF-κB activation is directly responsible for conferring an immune-evasive phenotype. We observed that NF-κB activation was associated with increased occupancy of the CD47 promoter, leading to a corresponding increase in CD47 surface abundance on ESCC cells following radiotherapy. Consequently, inhibiting NF-κB or blocking the CD47–SIRPα interaction with a specific antibody restored the phagocytic capacity of macrophages against these irradiated tumor cells.

Targeting the Cholinergic Loop with Engineered Bacteria Reverses Radioresistance and Enhances Therapeutic Efficacy

        Finally, we developed and tested a therapeutic strategy to radiosensitize ESCC by targeting this signaling nexus. While the nAChR antagonist mecamylamine showed efficacy, we designed a more targeted approach using an engineered tumor-tropic probiotic, E. coli Nissle 1917 (EcN), known for its preferential colonization of the hypoxic tumor microenvironment. These bacteria were engineered to function as an "ACh scavenger" by releasing a bacterially-derived cholinesterase-like enzyme (ChoE), which effectively cleared the RT-induced surge of ACh in the tumor milieu. This intervention successfully dampened Ca²⁺ peaks and reduced NF-κB and β-catenin activity, consequently suppressing both EMT and immune evasion.

        When combined with RT, this strategy showed significant synergistic effects. RT enhanced bacterial colonization and permeability, while the engineered bacteria, by depleting ACh, dismantled the α3β4-Ca²⁺-NF-κB-CD47 axis. This combination not only cleared residual tumor cells more effectively but also mitigated the risk of immune escape and metastatic recurrence often associated with conventional radiotherapy.

        In conclusion, we propose a novel mechanism driving radioresistance in ESCC centered on a self-reinforcing cholinergic signaling loop. We demonstrate that radiotherapy initiates an NF-κB-centric vicious cycle: Radiation → ROS → NF-κB activation → ChAT-driven ACh synthesis → α3β4 receptor-mediated Ca²⁺ influx, which in turn further amplifies NF-κB activity. This feedback loop promotes malignant progression and post-radiotherapy recurrence through two key downstream effects. Sustained Ca²⁺ signaling induces EMT to enhance invasion, while amplified NF-κB upregulates the "don't eat me" signal CD47 to facilitate immune evasion from macrophages. This dual pro-invasive and anti-phagocytic phenotype provides a robust biological rationale for the poor prognosis linked to high CHRNB4 expression in ESCC.

         More importantly, the elucidation of this mechanism identifies highly promising therapeutic targets for overcoming ESCC radioresistance. Conventional drugs, such as the nicotinic receptor antagonist mecamylamine, could potentially be used to disrupt this loop. However, our proposed synthetic biology-based, tumor-tropic engineered bacteria serving as an "ACh sink" represent a more precise and innovative interventional strategy. By specifically degrading ACh within the tumor microenvironment, this therapy is poised to dismantle the entire malignant signaling network at its source, thereby restoring tumor cell radiosensitivity and reversing their immune-evasive status. We believe that targeting this "NF-κB–cholinergic–CD47" signaling axis, particularly in combination with radiotherapy and immunotherapy, will open a new avenue for improving therapeutic outcomes for ESCC patients.