Cancer target discovery for biomarker development, imaging and radionuclide loaded nanoparticle therapy

Abstract

Background 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 (300 mm3) and large (700 mm3) 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

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Tim Smith (Supervisor), Catharine West (Supervisor) & Ananya Choudhury (Supervisor)