Choudhury, AnanyaWest, CatharineSmith, TimShabbir, Rekaya2025-06-252023-12-04https://hdl.handle.net/20.500.14154/75669Background and aims: Patients with hypoxic muscle invasive bladder cancer (MIBC) have a poor prognosis and overall survival (OS) rate. There is a need to develop biomarkers for informing on hypoxia-targeting therapy. Gene expression signatures can predict benefit from hypoxia modification to improve outcome. Imaging genomics links medical images with molecular profiling to discover imaging biomarkers that could reflect hypoxia. Molecular radiotherapy (MRT) targets specific receptors expressed by cells. The overexpression of EGFR also associates with poor survival rates, EGFR inhibitors are promising but tumour heterogeneity is problematic. The thesis aims were to: 1) identify genes upregulated by hypoxia in bladder cancer cells; 2) investigate whether the West 24-gene bladder hypoxia signature is sensitive to changes in oxygen levels; 3) use MRI to identify hypoxia in large and small tumours in vivo; 4) identify new hypoxia-associated gene panels from transcriptomic data generated in vitro and in vivo; 5) identify bladder cancer hypoxic biomarkers and surface membrane targets for MRT using proteomics in MIBC; and 6) study the uptake and dose-distributions of EGFR-targeted 177Lu or 90Y radiolabelled-AuNPs. Methods: 1) Six BC cell lines (HT1376, T24, J82, UMUC3, RT4, RT112) were exposed to normoxia (21% O2) and hypoxia (1%, 0.1% and 0.2% O2) for 24h. RNA was extracted, transcriptomic data generated using Clariom S Microarrays and expression of hypoxia upregulated genes identified. 2) The data were used to explore changes in West 24-gene signature scores. 3) Small (300mm3) and large (700mm3) xenografts were established for HT1376 MIBC cells. Hypoxia was identified using pimonidazole (PIMO), OE-MRI and DCE-MRI. Differential gene expression was determined. 4) Gene panels were derived from in vitro and in vivo transcriptomic data and tested (log-rank Mantel Cox test) in a TCGA bladder cancer cohort (n=412). Hypoxia scores were generated using the median expression of the genes in a signature/panel. 5) HT1376, T24 and J82 cells were cultured in normoxia (21% O2) and hypoxia (1% and 0.1% O2) for 24h and 48h. Proteins were extracted and analysed using LC-MS and SWATH-MS, and the data used to identify surface membrane targets. Differential expression of EGFR and hypoxia markers (CAIX, GLUT-1) was measured using transcriptomics, proteomics, western blot and EGFR-ELISA. 6) Anti-EGFR conjugated radiolabelled (177Lu or 90Y) AuNPs were used to study dose distributions in vivo. Results: 1) 77 genes were significantly upregulated (padj≤ 0.001) in hypoxia (0.1% O2) across ≥3 cell lines. Three genes (DPYSL2, SYDE1, SLC2A3) were in both the West 24-gene signature and new 77-gene panel. 2) The expression of the 24-gene West signature increased with decreasing O2 levels. 3) Hypoxic regions were identified in small and large xenografted tumours using a combination of OE-MRI and DCE-MRI approaches. The in vivo transcriptomics analysis identified gene expression differences between PIMO-high(hypoxic) and PIMO-low(normoxic) regions and differences in HSs generated from the 24-gene signature (p<0.0052) and the new 77-gene panel (p<0.0025). 4) Gene signatures/panels were prognostic for overall survival: 24-gene (p<0.000064), 77-gene (p<0.01), 3 common genes (p<0.0013). 5) 26 proteins had consistently higher expression in hypoxia across all three cell lines. A gene panel based on the 26 proteins was prognostic in the TCGA cohort (p<0.00065). Eleven plasma membrane proteins were identified as upregulated under hypoxia. No significant differential expression of EGFR was seen in BC cells by hypoxia. The expression of hypoxia markers (CAIX and GLUT-1) was significantly increased by hypoxia using different measurement approaches in all bladder cells. 6) The coefficient of variation of EGFR-targeted 90Y-radiolabelled-AuNPs was less than EGFR-targeted 177Lu-radiolabelled-AuNPs in xenografted tumours. Conclusions: 1) Hypoxia influences the expression of many common genes across different bladder cancer cell lines. 2) The West 24-gene bladder hypoxia score is sensitive to changes in O2 levels in vitro showing it reflects differences in hypoxia. 3) Gene panels derived from hypoxic cells in vitro inform on hypoxia and prognosis 4) The West 24-gene signature performed best as a biomarker of hypoxia. 5) Proteomic profiling identified cell membrane markers to study for MRT. 6) Anti-EGFR radionuclides labelled AuNPs provided insight about the heterogeneity and dosimetry of MRT.245enCancerMRIMRTRadionuclide ThrapyTargeted TherapyBiomarker discoveryHypoxiaEGFRThesis