Kaye J. Williams

Background: Radiotherapy induces systemic changes beyond the targeted tumour site, yet the biological mechanisms driving these effects remain poorly understood. This study aims to longitudinally profile acute systemic plasma proteomic responses to radiotherapy in patients with prostate, bladder, and head and neck cancers, providing insight into shared and tumour-specific effects and identifying biomarkers predictive of treatment-related toxicities. Methods: We apply our previously developed Nano-proteomics workflow to comprehensively analyse longitudinal weekly plasma samples collected before and during radiotherapy. We perform mass spectrometry-based differential protein analysis to identify acute changes of the plasma proteome and enriched biological pathways involved. In the prostate cohort, we associate plasma proteomics with clinical toxicity outcomes. Results: Our data indicate that the most significant systemic proteomic changes occur within the first two weeks of radiotherapy, highlighting a critical period for biomarker identification. Across all patient cohorts, we observe common biological responses: rapid activation of inflammatory and immune pathways, followed by structural reorganisation and immune resolution, regardless of tumour type or concurrent treatments. Despite this shared acute response, distinct protein mediators are found to be dysregulated in a tumour-specific manner. In the prostate cancer cohort, plasma profiling at baseline, one week after the initiation of radiotherapy, and at the end of radiotherapy, results in the identification of 28, 29, and 20 proteins, respectively, that are associated with subsequent bowel and urinary toxicities. Conclusions: This study underscores the value of longitudinal proteomics in uncovering systemic effects of radiotherapy and supports the potential of plasma proteomic biomarkers to identify patients at increased risk of radiotherapy-induced toxicity, paving the way for personalised treatment strategies.

Supplementary Figure S5 compares the natural history growth of Calu6 and U87 xenografts.

Supplementary Figure S6 shows that banoxantrone induces tumor necrosis.

Supplementary Figure S1 shows a summary of the cohorts of xenografts used in each experiment.

Supplementary Figure S4 confirms that there was no drug effect on cell number or viability.

Supplementary Figure S3 shows the MRI analysis steps.

Supplementary Figure S2: Confirmation that oxygen increases tumor R1.

Oxygen-enhanced MRI (OE-MRI) has shown promise for quantifying and spatially mapping tumor hypoxia, either alone or in combination with perfusion imaging. Previous studies have validated the technique in mouse models and in patients with cancer. Here, we report the first evidence that OE-MRI can track change in tumor oxygenation induced by two drugs designed to modify hypoxia. Mechanism of action of banoxantrone and atovaquone were confirmed using in vitro experiments. Next, in vivo OE-MRI studies were performed in Calu6 and U87 xenograft tumor models, alongside fluorine-18–fluoroazomycin arabinoside PET and immunohistochemistry assays of hypoxia. Neither drug altered tumor size. Banoxantrone reduced OE-MRI hypoxic fraction in Calu6 tumors by 52.5% ± 12.0% (P = 0.008) and in U87 tumors by 29.0% ± 15.8% (P = 0.004) after 3 days treatment. Atovaquone reduced OE-MRI hypoxic fraction in Calu6 tumors by 53.4% ± 15.3% (P = 0.002) after 7 days therapy. PET and immunohistochemistry provided independent validation of the MRI findings. Finally, combined OE-MRI and perfusion imaging showed that hypoxic tissue was converted into necrotic tissue when treated by the hypoxia-activated cytotoxic prodrug banoxantrone, whereas hypoxic tissue became normoxic when treated by atovaquone, an inhibitor of mitochondrial complex III of the electron transport chain. OE-MRI detected and quantified hypoxia reduction induced by two hypoxia-modifying therapies and could distinguish between their differential mechanisms of action. These data support clinical translation of OE-MRI biomarkers in clinical trials of hypoxia-modifying agents to identify patients demonstrating biological response and to optimize treatment timing and scheduling.Significance:For the first time, we show that hypoxic fraction measured by oxygen-enhanced MRI (OE-MRI) detected changes in tumor oxygenation induced by two drugs designed specifically to modify hypoxia. Furthermore, when combined with perfusion imaging, OE-MRI hypoxic volume distinguished the two drug mechanisms of action. This imaging technology has potential to facilitate drug development, enrich clinical trial design, and accelerate clinical translation of novel therapeutics into clinical use.

Supplementary Figure S6 shows that banoxantrone induces tumor necrosis.