Presenter(s)
M. Yuan1,2, W. Cui3,4, R. Zhang5, D. Chen6, and J. Yu6; 1Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 2Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 3School of Clinical Medicine, Shandong Second Medical University, Weifang, China, 4Department of Radiation Oncology and Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, 5Shandong University Cancer Center;Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, 6Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China
Purpose/Objective(s):
Despite the considerable potential of combining immunotherapy with radiotherapy (iRT) in the treatment of NSCLC, approximately 60% of patients do not benefit from this approach. Radiotherapy can remodel cancer-associated fibroblasts (CAFs), enhancing their immunosuppressive properties, which is a critical factor leading to immune escape. This study aims to investigate the molecular mechanism by which N-glycosylation of ecto-5'-nucleotidase (NT5E) in CAFs mediates radio-immunotherapy resistance in NSCLC and to explore potential strategies.Materials/Methods:
Multi-omics analysis of radiotherapy-resistant CAFs and clinical samples identified NT5E as a key glycosylated target.In vitro validation was conducted using WB and IF in irradiated CAFs. The functional role of NT5E N-glycosylation was assessed in transplantation tumor models using immunodeficient and immunocompetent mice, employing NT5E glycosylation site mutation and the N-glycosylation inhibitor tunicamycin (TM). The involvement of CD8+ T cells was confirmed through in vivo depletion experiments and in vitro co-culture systems (fibroblasts, tumor cells, T cells).Co-IP-MS identified RPN1 as an upstream regulator; downstream mechanisms were elucidated via RNA-seq, secretomics, and adenosine assays. Finally, fibroblast-specific Rpn1 knockout and humanized mouse models demonstrated that targeting this axis overcomes immune checkpoint blockade resistance.Results: Comparing tumor tissues from NSCLC patients who responded to radiotherapy versus those who did not, higher NT5E N-glycosylation levels in CAFs were associated with poor response. Mechanistically, radiotherapy significantly upregulated NT5E N-glycosylation levels in CAFs. Either mutating the NT5E N-glycosylation site or inhibiting its glycosylation, consequently enhancing CAF activation. As an upstream regulator, RPN1 facilitated the membrane translocation of NT5E. Glycosylated NT5E promoted the breakdown of extracellular ATP into immunosuppressive adenosine, which remodeled the tumor immune microenvironment by inhibiting CD8+ T cell function. Correspondingly, inhibiting RPN1 effectively reduced NT5E N-glycosylation levels in CAFs, which not only enhanced the sensitivity of NSCLC to radiotherapy but also synergistically overcame ICB resistance in humanized mouse models. Importantly, multiplex immunofluorescence staining of patient tumor tissues revealed that iRT-responsive tumors exhibited significantly reduced NT5E N-glycosylation in CAFs and increased infiltration of CD8+ T cells compared to non-responsive tumors.
Conclusion: This study elucidates a novel mechanism by which RPN1 regulates NT5E N-glycosylation to influence adenosine metabolism and remodel the immune microenvironment. Clinical evidence positions CAF-specific NT5E N-glycosylation as a potential predictive biomarker and therapeutic target to overcome radiotherapy resistance and optimize the immune microenvironment in NSCLC.