S2556
Radiobiology - Microenvironment
ESTRO 2026
neighbour (KNN) density estimation to identify hypoxia-associated neutrophil clusters. Prognostic capacity ofhypoxia-associatedneutrophil markers was assessed in the TCGA Bladder and BCON cohorts via Kaplan-Meier. Results: Flow cytometry revealed two distinct neutrophil clusters associated with normoxic and hypoxic tumour-secreted factors. Cluster 1 (CD66b+ICAM1+) was significantly enriched in 21% O2 (38.28%), versus 1% O2 (12.26%, t = 3.931, p = 0.0171)and 0.2% O2 (6.193%, t = 4.902, p = 0.0080). Cluster 2 (CD66b+ICAM1+CD62L+) was significantly enriched in 1% (30.73%, t = 5.734, p = 0.0056) and 0.2% O2 (19.97%, t = 3.207, p = 0.0184) compared to 21% O2 secreted factors (9.093%).
randomised clinical trial. BMJ (Clinical research ed.), 370, m2836. 2.Kulcenty, K. et al. (2019). The Composition of Surgical Wound Fluids from Breast Cancer Patients is Affected by Intraoperative Radiotherapy Treatment and Depends on the Molecular Subtype of Breast Cancer. Cancers, 12(1), 11 Keywords: CAFs, intraoperative radiotherapy
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The interplay between the hypoxic tumour microenvironment and neutrophils in bladder cancer radiotherapy resistance Fraser B Child 1 , Conrado G Quiles 1 , Sapna Lunj 1 , Luisa V Biolatti 1 , Eleanor J Cheadle 1 , Vodathi Bamunuarachchi 2 , Julie Gough 3 , Martin J Humphries 4 , kimberley reeves 1 , Peter J Hoskin 2,1 , Ananya Choudhury 2,1 1 1Division of Cancer Sciences, University of Manchester, Manchester, United Kingdom. 2 The Christie NHS Foundation Trust, NHS, Manchester, United Kingdom. 3 2Department of Materials & Henry Royce Institute, University of Manchester, Manchester, United Kingdom. 4 3Manchester Cell-Matrix Centre, Division of Cell Matrix Biology & Regenerative Medicine, University of Manchester, Manchester, United Kingdom Purpose/Objective: Hypoxia is a well-established poor prognostic factor associated with radioresistance1. One mechanism of hypoxia-induced radioresistance is neutrophil- extracellular trap-mediated extracellular matrix (ECM) remodelling, which promotes pro-tumour immune cell exclusion. Hypoxic ECM remodelling polarises tumour- infiltrated neutrophils towards pro-tumour phenotypes2. Neutrophil infiltration is an independent adverse prognostic factor linked with poor radiotherapy responses 3. A high intratumoural neutrophil:CD8+ T cell ratio associates with poor prognosis following radiotherapy 4. Despite this evidence, the mechanisms driving radiotherapy resistance are not completely understood. We aimed to investigate the interplay between hypoxia- associated cell-secreted factors on neutrophil polarisation and their association with outcomes in muscle-invasive bladder cancer (MIBC). Material/Methods: T24 MIBC cells were incubated in 21%, 1% and 0.2% oxygen and media were collected after 7 days. Neutrophils were isolated from the whole blood of healthy donors (n=3) and incubated in normoxic (21% O2)and hypoxic (1%, 0.2% O2) T24-conditioned media. Neutrophil viability and phenotyping were analysed by flow cytometry after 24 hours using t-distributed stochastic neighbour embedding (t-SNE) and K-nearest
While Kaplan-Meier demonstrated ICAM1 expression was not prognostic in BCON, in the TCGA cohort, patients with medium (33 – 65%) ICAM1 scores had the poorest 10-year overall survival, followed by high ( ≥ 66%) scores when compared to low ( ≤ 32%) ICAM1 scores (p = 0.019).
Conclusion: Hypoxia-induced soluble factors from T24 cells drive neutrophil ICAM1 and CD62L expression in vitro. ICAM1 expression directly affects patient outcome in a surgery-treated cohort, suggesting potential predictive value across treatments; however, this warrants a comprehensive evaluation. The role of ICAM1+ neutrophils in radiotherapy resistance needs further characterisation. Future transcriptomic analyses should define the molecular pathways involved in neutrophil reprogramming during hypoxia. An improved understanding of these mechanisms will unveil novel targets to modulate immune response
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