A plasma derived hypoxia signature predicts benefit from hypoxia-modifying radiotherapy and reveals dynamic humoral immune modulation in bladder cancer.

INTRODUCTION: Hypoxia is a hallmark of solid tumours and drives radioresistance, yet no hypoxia biomarkers are used clinically. We explored nanoproteomics to identify a plasma protein signature for longitudinal non-invasive hypoxia monitoring. METHODS: Twenty-three bladder cancer patients (T2-T3b) undergoing radiotherapy (55 Gy, 4 weeks) were prospectively enroled. Plasma was collected at baseline and weekly, nanoparticle-enriched and analysed by proteomics. Hypoxia scores (HS) were derived from diagnostic biopsies using a validated 24-gene signature, and patients stratified by median HS. RESULTS: We identified 115 differentially abundant proteins (DAPs; |FC | > 1.5; p < 0.05). Seven DAPs (IGKV3, IGLV2-18, VL_4, VL_7, FN1, IGLV2-14, CAMP) correlated with HS across multiple timepoints (p < 0.05; |r | >0.4). A two-protein signature (FN1, CAMP) was retrospectively validated, showing prognostic value in TCGA-BLCA (n = 404; HR = 1.54; CI = 1.12-2.13; p = 0.009), BC2001 (n = 313; HR = 1.42; CI = 1.08-1.86; p = 0.011) and in meta-analysis (n = 150; HR = 1.48; CI = 1.23-1.78; p < 0.001). In BCON (n = 150), it predicted benefit from hypoxia-modifying therapy (HR = 0.60; CI = 0.34-1.06; p = 0.079). The 115 DAPs formed five temporal co-expression clusters peaking at successive treatment weeks. Clusters were enriched for humoral immune pathways, with one linked to extracellular matrix remodelling. CONCLUSION: These findings support the use of low abundance plasma biomarkers for real-time hypoxia stratification, revealing dynamic immune responses during radiotherapy associated with hypoxia.