The study enrolled 23 patients with muscle-invasive bladder cancer, stages T2 to T3b, who were undergoing radical radiotherapy delivered over four weeks alongside weekly gemcitabine chemotherapy. Blood samples were collected before the first radiation fraction and then every week until treatment ended. The researchers also retrieved each patient’s diagnostic biopsy and measured the activity of a validated 24-gene hypoxia signature, generating a hypoxia score for every tumour. Patients were then split into high and low hypoxia groups based on the median score. This design allowed the team to ask a deceptively simple question: do the proteins circulating in the blood of patients with oxygen-starved tumours look different from those in patients with well-oxygenated tumours, and do they change as treatment progresses?

Answering that question required overcoming a long-standing technical obstacle. Plasma is dominated by a small number of extremely abundant proteins, such as albumin and immunoglobulins, which mask the low-abundance molecules that often carry the most informative biological signals. Previous attempts to find circulating hypoxia markers relied on targeted antibody tests of single proteins, and several promising candidates, including osteopontin in head and neck cancer, ultimately failed prospective validation. The Manchester team took a different approach, using engineered liposomal nanoparticles roughly 105 nanometres in diameter as molecular fishing hooks. When incubated with plasma, these particles attract a shell of bound proteins, known as a protein corona, which preferentially captures lower-abundance molecules. The corona proteins were then purified, digested and analysed by high-resolution liquid chromatography tandem mass spectrometry.

The results were striking. Across all time points, the analysis identified 816 plasma proteins, of which 115 showed significant differences between patients with high and low tumour hypoxia scores, defined as a fold change greater than 1.5 and a p-value below 0.05. The number of differentially abundant proteins fluctuated across the treatment timeline: 24 before radiotherapy, 46 after one week, 40 after two weeks, 14 after three weeks and 37 at the end of treatment. Remarkably, roughly 45 percent of the differentially abundant proteins were immunoglobulins or complement cascade proteins, pointing to a previously underappreciated connection between tumour hypoxia and the humoral immune system, the antibody-producing arm of immunity.

From the full set of hypoxia-associated proteins, seven candidates stood out because they correlated significantly with tumour hypoxia scores at two or more time points and maintained a consistent direction of correlation throughout treatment. Five of them, IGKV3, IGLV2-18, VL_4 and VL_7 alongside the extracellular matrix protein FN1, were consistently elevated in patients with hypoxic tumours, while IGLV2-14 and CAMP were consistently reduced. Principal component analysis and K-means clustering showed that this seven-protein signature cleanly separated high-hypoxia from low-hypoxia patients, and, crucially, that the separation held steady at every time point during radiotherapy. That temporal stability is exactly what a clinically useful monitoring biomarker requires, since a signal that drifts unpredictably during treatment cannot guide real-time decisions.

Because only two of the seven proteins, FN1 and CAMP, had matching RNA expression data in public cohorts, the team distilled the signature into a two-gene version and validated it retrospectively in three independent bladder cancer cohorts. In the TCGA-BLCA cohort of 404 patients treated with cystectomy, high hypoxia scores predicted poorer ten-year overall survival with a hazard ratio of 1.54. In the BC2001 radiotherapy cohort of 313 patients, the hazard ratio was 1.42. A meta-analysis combining TCGA-BLCA, BC2001 and the BCON trial confirmed the association with poor prognosis across 150 patients, with a hazard ratio of 1.48, independently of age, sex, stage and treatment received. The signature also remained prognostic in patients treated with and without concurrent chemotherapy, suggesting its signal is not an artefact of a particular drug regimen.

The most clinically provocative finding came from the BCON phase III trial, which tested whether breathing carbogen, a mixture of oxygen and carbon dioxide, together with nicotinamide, a drug that improves blood flow, could sensitise hypoxic tumours to radiation. That trial previously showed a 17 percent improvement in five-year overall survival with hypoxia modification. When the new two-gene signature was applied to BCON samples, patients with high hypoxia scores had poorer survival in the radiotherapy-only arm but lost that disadvantage when treated with carbogen and nicotinamide, with a hazard ratio of 0.60 favouring hypoxia modification. In other words, the blood-based signature did not merely forecast prognosis; it predicted who would actually benefit from targeting hypoxia, mirroring the performance of the original 24-gene tumour biopsy signature but requiring only a blood draw.

Beyond the biomarker itself, the study revealed a dynamic biological story. Weighted correlation analysis grouped the 115 hypoxia-associated proteins into five co-expression clusters, each peaking at a successive point in the treatment timeline: before radiotherapy, and at weeks one through four. Every cluster was enriched for B cell response and immunoglobulin production pathways, while the pre-treatment cluster was uniquely enriched for extracellular matrix remodelling, and later clusters for coagulation, fibrin formation and metabolic processes. Comparing expression patterns with fold-change dynamics suggested that radiotherapy primarily drove the timing of cluster activation, whereas tumour hypoxia shaped the baseline abundance and magnitude of the response. The researchers propose that tumour re-oxygenation during radiotherapy, which generates reactive oxygen species and activates HIF-1 signalling, may explain the declining protein levels seen in later weeks of treatment.

The immunological implications extend well beyond bladder cancer. Prior laboratory work has shown that hypoxia-inducible factors directly regulate B cell maturation and immunoglobulin class switching, and mouse studies have demonstrated that germinal centre hypoxia can impair humoral anti-tumour responses. This study provides the first clinical, plasma-level evidence that such immune modulation occurs in patients, with hypoxic tumours showing a shifted immunoglobulin profile that evolves over the course of radiotherapy. The authors caution that the discovery cohort was small, included only two female patients, and lacked direct protein-level outcome validation, so prospective validation at the protein level is the essential next step. Even so, the work establishes nanoparticle-enabled proteomics as a powerful discovery platform for circulating cancer biomarkers and suggests a future in which a routine blood test could tell oncologists, week by week, whether a patient’s tumour is suffocating, whether radiation is working, and whether hypoxia-targeting drugs should be added to the plan.

Subject of Research: A plasma protein signature for detecting tumour hypoxia and predicting benefit from hypoxia-modifying radiotherapy in bladder cancer

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

Article References: Guerrero Quiles, C., Abumanhal-Masarweh, H., G. Abalos, J., Lodhi, T., Sanchez-Martinez, D., Reeves, K., James, N. D., Hall, E., Huddart, R. A., Porta, N., Hoskin, P., Biolatti, L. V., Hadjidemetriou, M., West, C. M., & Choudhury, A. (2026). A plasma derived hypoxia signature predicts benefit from hypoxia-modifying radiotherapy and reveals dynamic humoral immune modulation in bladder cancer. British Journal of Cancer. https://doi.org/10.1038/s41416-026-03603-x

Image Credits: AI Generated

DOI: 10.1038/s41416-026-03603-x

Keywords: bladder cancer, tumour hypoxia, radiotherapy, biomarker, proteomics, nanoparticles, protein corona, mass spectrometry, humoral immunity, BCON trial, carbogen and nicotinamide, liquid biopsy