ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The Estrogen Receptor-alpha (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 hours. GSEA analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumours cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Enabling large-scale screening of Barrett's esophagus using weakly supervised deep learning in histopathology.

Timely detection of Barrett's esophagus, the pre-malignant condition of esophageal adenocarcinoma, can improve patient survival rates. The Cytosponge-TFF3 test, a non-endoscopic minimally invasive procedure, has been used for diagnosing intestinal metaplasia in Barrett's. However, it depends on pathologist's assessment of two slides stained with H&E and the immunohistochemical biomarker TFF3. This resource-intensive clinical workflow limits large-scale screening in the at-risk population. To improve screening capacity, we propose a deep learning approach for detecting Barrett's from routinely stained H&E slides. The approach solely relies on diagnostic labels, eliminating the need for expensive localized expert annotations. We train and independently validate our approach on two clinical trial datasets, totaling 1866 patients. We achieve 91.4% and 87.3% AUROCs on discovery and external test datasets for the H&E model, comparable to the TFF3 model. Our proposed semi-automated clinical workflow can reduce pathologists' workload to 48% without sacrificing diagnostic performance, enabling pathologists to prioritize high risk cases.

SliDL: A toolbox for processing whole-slide images in deep learning.

The inspection of stained tissue slides by pathologists is essential for the early detection, diagnosis and monitoring of disease. Recently, deep learning methods for the analysis of whole-slide images (WSIs) have shown excellent performance on these tasks, and have the potential to substantially reduce the workload of pathologists. However, WSIs present a number of unique challenges for analysis, requiring special consideration of image annotations, slide and image artefacts, and evaluation of WSI-trained model performance. Here we introduce SliDL, a Python library for performing pre- and post-processing of WSIs. SliDL makes WSI data handling easy, allowing users to perform essential processing tasks in a few simple lines of code, bridging the gap between standard image analysis and WSI analysis. We introduce each of the main functionalities within SliDL: from annotation and tile extraction to tissue detection and model evaluation. We also provide 'code snippets' to guide the user in running SliDL. SliDL has been designed to interact with PyTorch, one of the most widely used deep learning libraries, allowing seamless integration into deep learning workflows. By providing a framework in which deep learning methods for WSI analysis can be developed and applied, SliDL aims to increase the accessibility of an important application of deep learning.

Lesion-specific 3D-printed moulds for image-guided tissue multi-sampling of ovarian tumours: A prospective pilot study.

Background: High-Grade Serous Ovarian Carcinoma (HGSOC) is the most prevalent and lethal subtype of ovarian cancer, but has a paucity of clinically-actionable biomarkers due to high degrees of multi-level heterogeneity. Radiogenomics markers have the potential to improve prediction of patient outcome and treatment response, but require accurate multimodal spatial registration between radiological imaging and histopathological tissue samples. Previously published co-registration work has not taken into account the anatomical, biological and clinical diversity of ovarian tumours. Methods: In this work, we developed a research pathway and an automated computational pipeline to produce lesion-specific three-dimensional (3D) printed moulds based on preoperative cross-sectional CT or MRI of pelvic lesions. Moulds were designed to allow tumour slicing in the anatomical axial plane to facilitate detailed spatial correlation of imaging and tissue-derived data. Code and design adaptations were made following each pilot case through an iterative refinement process. Results: Five patients with confirmed or suspected HGSOC who underwent debulking surgery between April and December 2021 were included in this prospective study. Tumour moulds were designed and 3D-printed for seven pelvic lesions, covering a range of tumour volumes (7 to 133 cm3) and compositions (cystic and solid proportions). The pilot cases informed innovations to improve specimen and subsequent slice orientation, through the use of 3D-printed tumour replicas and incorporation of a slice orientation slit in the mould design, respectively. The overall research pathway was compatible with implementation within the clinically determined timeframe and treatment pathway for each case, involving multidisciplinary clinical professionals from Radiology, Surgery, Oncology and Histopathology Departments. Conclusions: We developed and refined a computational pipeline that can model lesion-specific 3D-printed moulds from preoperative imaging for a variety of pelvic tumours. This framework can be used to guide comprehensive multi-sampling of tumour resection specimens.

Computational pathology aids derivation of microRNA biomarker signals from Cytosponge samples.

BACKGROUND: Non-endoscopic cell collection devices combined with biomarkers can detect Barrett's intestinal metaplasia and early oesophageal cancer. However, assays performed on multi-cellular samples lose information about the cell source of the biomarker signal. This cross-sectional study examines whether a bespoke artificial intelligence-based computational pathology tool could ascertain the cellular origin of microRNA biomarkers, to inform interpretation of the disease pathology, and confirm biomarker validity. METHODS: The microRNA expression profiles of 110 targets were assessed with a custom multiplexed panel in a cohort of 117 individuals with reflux that took a Cytosponge test. A computational pathology tool quantified the amount of columnar epithelium present in pathology slides, and results were correlated with microRNA signals. An independent cohort of 139 Cytosponges, each from an individual patient, was used to validate the findings via qPCR. FINDINGS: Seventeen microRNAs are upregulated in BE compared to healthy squamous epithelia, of which 13 remain upregulated in dysplasia. A pathway enrichment analysis confirmed association to neoplastic and cell cycle regulation processes. Ten microRNAs positively correlated with columnar epithelium content, with miRNA-192-5p and -194-5p accurately detecting the presence of gastric cells (AUC 0.97 and 0.95). In contrast, miR-196a-5p is confirmed as a specific BE marker. INTERPRETATION: Computational pathology tools aid accurate cellular attribution of molecular signals. This innovative design with multiplex microRNA coupled with artificial intelligence has led to discovery of a quality control metric suitable for large scale application of the Cytosponge. Similar approaches could aid optimal interpretation of biomarkers for clinical use. FUNDING: Funded by the NIHR Cambridge Biomedical Research Centre, the Medical Research Council, the Rosetrees and Stoneygate Trusts, and CRUK core grants.

Hyperpolarized 13C-Pyruvate Metabolism as a Surrogate for Tumor Grade and Poor Outcome in Renal Cell Carcinoma-A Proof of Principle Study.

Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1-13C]pyruvate (HP-13C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP-13C-MRI and conventional proton (1H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant (kPL) between 13C-pyruvate and 13C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing kPL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset (p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP-13C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP-13C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.

Triage-driven diagnosis of Barrett's esophagus for early detection of esophageal adenocarcinoma using deep learning.

Deep learning methods have been shown to achieve excellent performance on diagnostic tasks, but how to optimally combine them with expert knowledge and existing clinical decision pathways is still an open challenge. This question is particularly important for the early detection of cancer, where high-volume workflows may benefit from (semi-)automated analysis. Here we present a deep learning framework to analyze samples of the Cytosponge-TFF3 test, a minimally invasive alternative to endoscopy, for detecting Barrett's esophagus, which is the main precursor of esophageal adenocarcinoma. We trained and independently validated the framework on data from two clinical trials, analyzing a combined total of 4,662 pathology slides from 2,331 patients. Our approach exploits decision patterns of gastrointestinal pathologists to define eight triage classes of varying priority for manual expert review. By substituting manual review with automated review in low-priority classes, we can reduce pathologist workload by 57% while matching the diagnostic performance of experienced pathologists.

Investigating the relationship between diffusion kurtosis tensor imaging (DKTI) and histology within the normal human brain.

Measurements of water diffusion with MRI have been used as a biomarker of tissue microstructure and heterogeneity. In this study, diffusion kurtosis tensor imaging (DKTI) of the brain was undertaken in 10 healthy volunteers at a clinical field strength of 3 T. Diffusion and kurtosis metrics were measured in regions-of-interest on the resulting maps and compared with quantitative analysis of normal post-mortem tissue histology from separate age-matched donors. White matter regions showed low diffusion (0.60 ± 0.04 × 10-3 mm2/s) and high kurtosis (1.17 ± 0.06), consistent with a structured heterogeneous environment comprising parallel neuronal fibres. Grey matter showed intermediate diffusion (0.80 ± 0.02 × 10-3 mm2/s) and kurtosis (0.82 ± 0.05) values. An important finding is that the subcortical regions investigated (thalamus, caudate and putamen) showed similar diffusion and kurtosis properties to white matter. Histological staining of the subcortical nuclei demonstrated that the predominant grey matter was permeated by small white matter bundles, which could account for the similar kurtosis to white matter. Quantitative histological analysis demonstrated higher mean tissue kurtosis and vector standard deviation values for white matter (1.08 and 0.81) compared to the subcortical regions (0.34 and 0.59). Mean diffusion on DKTI was positively correlated with tissue kurtosis (r = 0.82, p < 0.05) and negatively correlated with vector standard deviation (r = -0.69, p < 0.05). This study demonstrates how DKTI can be used to study regional structural variations in the cerebral tissue microenvironment and could be used to probe microstructural changes within diseased tissue in the future.

Cytosponge-trefoil factor 3 versus usual care to identify Barrett's oesophagus in a primary care setting: a multicentre, pragmatic, randomised controlled trial.

BACKGROUND: Treatment of dysplastic Barrett's oesophagus prevents progression to adenocarcinoma; however, the optimal diagnostic strategy for Barrett's oesophagus is unclear. The Cytosponge-trefoil factor 3 (TFF3) is a non-endoscopic test for Barrett's oesophagus. The aim of this study was to investigate whether offering this test to patients on medication for gastro-oesophageal reflux would increase the detection of Barrett's oesophagus compared with standard management. METHODS: This multicentre, pragmatic, randomised controlled trial was done in 109 socio-demographically diverse general practice clinics in England. Randomisation was done both at the general practice clinic level (cluster randomisation) and at the individual patient level, and the results for each type of randomisation were analysed separately before being combined. Patients were eligible if they were aged 50 years or older, had been taking acid-suppressants for symptoms of gastro-oesophageal reflux for more than 6 months, and had not undergone an endoscopy procedure within the past 5 years. General practice clinics were selected by the local clinical research network and invited to participate in the trial. For cluster randomisation, clinics were randomly assigned (1:1) by the trial statistician using a computer-generated randomisation sequence; for individual patient-level randomisation, patients were randomly assigned (1:1) by the general practice clinics using a centrally prepared computer-generated randomisation sequence. After randomisation, participants received either standard management of gastro-oesophageal reflux (usual care group), in which participants only received an endoscopy if required by their general practitioner, or usual care plus an offer of the Cytosponge-TFF3 procedure, with a subsequent endoscopy if the procedure identified TFF3-positive cells (intervention group). The primary outcome was the diagnosis of Barrett's oesophagus at 12 months after enrolment, expressed as a rate per 1000 person-years, in all participants in the intervention group (regardless of whether they had accepted the offer of the Cytosponge-TFF3 procedure) compared with all participants in the usual care group. Analyses were intention-to-treat. The trial is registered with the ISRCTN registry, ISRCTN68382401, and is completed. FINDINGS: Between March 20, 2017, and March 21, 2019, 113 general practice clinics were enrolled, but four clinics dropped out shortly after randomisation. Using an automated search of the electronic prescribing records of the remaining 109 clinics, we identified 13 657 eligible patients who were sent an introductory letter with 14 days to opt out. 13 514 of these patients were randomly assigned (per practice or at the individual patient level) to the usual care group (n=6531) or the intervention group (n=6983). Following randomisation, 149 (2%) of 6983 participants in the intervention group and 143 (2%) of 6531 participants in the usual care group, on further scrutiny, did not meet all eligibility criteria or withdrew from the study. Of the remaining 6834 participants in the intervention group, 2679 (39%) expressed an interest in undergoing the Cytosponge-TFF3 procedure. Of these, 1750 (65%) met all of the eligibility criteria on telephone screening and underwent the procedure. Most of these participants (1654 [95%]; median age 69 years) swallowed the Cytosponge successfully and produced a sample. 231 (3%) of 6834 participants had a positive Cytosponge-TFF3 result and were referred for an endoscopy. Patients who declined the offer of the Cytosponge-TFF3 procedure and all participants in the usual care group only had an endoscopy if deemed necessary by their general practitioner. During an average of 12 months of follow-up, 140 (2%) of 6834 participants in the intervention group and 13 (<1%) of 6388 participants in the usual care group were diagnosed with Barrett's oesophagus (absolute difference 18·3 per 1000 person-years [95% CI 14·8-21·8]; rate ratio adjusted for cluster randomisation 10·6 [95% CI 6·0-18·8], p<0·0001). Nine (<1%) of 6834 participants were diagnosed with dysplastic Barrett's oesophagus (n=4) or stage I oesophago-gastric cancer (n=5) in the intervention group, whereas no participants were diagnosed with dysplastic Barrett's oesophagus or stage I gastro-oesophageal junction cancer in the usual care group. Among 1654 participants in the intervention group who swallowed the Cytosponge device successfully, 221 (13%) underwent endoscopy after testing positive for TFF3 and 131 (8%, corresponding to 59% of those having an endoscopy) were diagnosed with Barrett's oesophagus or cancer. One patient had a detachment of the Cytosponge from the thread requiring endoscopic removal, and the most common side-effect was a sore throat in 63 (4%) of 1654 participants. INTERPRETATION: In patients with gastro-oesophageal reflux, the offer of Cytosponge-TFF3 testing results in improved detection of Barrett's oesophagus. Cytosponge-TFF3 testing could also lead to the diagnosis of treatable dysplasia and early cancer. This strategy will lead to additional endoscopies with some false positive results. FUNDING: Cancer Research UK, National Institute for Health Research, the UK National Health Service, Medtronic, and the Medical Research Council.

Three-Dimensional Printed Molds for Image-Guided Surgical Biopsies: An Open Source Computational Platform.

PURPOSE: Spatial heterogeneity of tumors is a major challenge in precision oncology. The relationship between molecular and imaging heterogeneity is still poorly understood because it relies on the accurate coregistration of medical images and tissue biopsies. Tumor molds can guide the localization of biopsies, but their creation is time consuming, technologically challenging, and difficult to interface with routine clinical practice. These hurdles have so far hindered the progress in the area of multiscale integration of tumor heterogeneity data. METHODS: We have developed an open-source computational framework to automatically produce patient-specific 3-dimensional-printed molds that can be used in the clinical setting. Our approach achieves accurate coregistration of sampling location between tissue and imaging, and integrates seamlessly with clinical, imaging, and pathology workflows. RESULTS: We applied our framework to patients with renal cancer undergoing radical nephrectomy. We created personalized molds for 6 patients, obtaining Dice similarity coefficients between imaging and tissue sections ranging from 0.86 to 0.96 for tumor regions and between 0.70 and 0.76 for healthy kidneys. The framework required minimal manual intervention, producing the final mold design in just minutes, while automatically taking into account clinical considerations such as a preference for specific cutting planes. CONCLUSION: Our work provides a robust and automated interface between imaging and tissue samples, enabling the development of clinical studies to probe tumor heterogeneity on multiple spatial scales.

Co-registration of optoacoustic tomography and magnetic resonance imaging data from murine tumour models.

As optoacoustic tomography (OT) emerges as a mainstream pre-clinical imaging modality, understanding the relationship between optoacoustic and other imaging biomarkers in the context of the underlying tissue biology becomes vitally important. Complementary insight into tumour vasculature and hypoxia can be gained using OT alongside magnetic resonance imaging (MRI)-based techniques. To evaluate the relationship between these metrics and the relative performance of the two modalities in assessment of tumour physiology, co-registration of their output imaging data is required. Unfortunately, this poses a significant challenge due to differences in animal positioning during imaging. Here, we present an integrated framework for registration of OT and MR image data in mice. Our framework combines a novel MR animal holder, to improve animal positioning during imaging, and a landmark-based software co-registration algorithm. We demonstrate that our protocol significantly improves registration of both body and tumour contours between these modalities, enabling more precise multi-modal tumour characterisation.

A multisample 7 T dynamic nuclear polarization polarizer for preclinical hyperpolarized MR.

Dynamic nuclear polarization (DNP) provides the opportunity to boost liquid state magnetic resonance (MR) signals from selected nuclear spins by several orders of magnitude. A cryostat running at a temperature of ~ 1 K and a superconducting magnet set to between 3 and 10 T are required to efficiently hyperpolarize nuclear spins. Several DNP polarizers have been implemented for the purpose of hyperpolarized MR and recent systems have been designed to avoid the need for user input of liquid cryogens. We herein present a zero boil-off DNP polarizer that operates at 1.35 ± 0.01 K and 7 T, and which can polarize two samples in parallel. The samples are cooled by a static helium bath thermally connected to a 1 K closed-cycle 4 He refrigerator. Using a modified version of the commercial fluid path developed for the SPINlab polarizer, we demonstrate that, within a 12-minute interval, the system can produce two separate hyperpolarized 13 C solutions. The 13 C liquid-state polarization of [1-13 C]pyruvate measured 26 seconds after dissolution was 36%, which can be extrapolated to a 55% solid state polarization. The system is well adapted for in vitro and in vivo preclinical hyperpolarized MR experiments and it can be modified to polarize up to four samples in parallel.

Role of TFF3 as an adjunct in the diagnosis of Barrett's esophagus using a minimally invasive esophageal sampling device-The CytospongeTM.

The incidence of esophageal carcinoma continues to increase whilst its prognosis remains poor. The most dramatic reduction in mortality is likely to follow early diagnosis of the preinvasive precursor lesion, Barrett's esophagus (BE), coupled with treatment of dysplastic lesions. The major risk factor for BE is gastroesophageal reflux disease, however this is highly prevalent and only a small proportion of individuals have BE, therefore an endoscopy-based screening strategy to detect BE is unfeasible. Minimally invasive esophageal sampling devices offer an alternative, cost-effective strategy which can be deployed within an at-risk population in a primary care setting to identify individuals with probable BE who can then be referred for endoscopic confirmation. The device that has currently progressed furthest in clinical trials is the CytospongeTM which collects cells from the gastric cardia, gastroesophageal junction and along the whole esophageal length. The cell sample is processed into a formalin-fixed paraffin-embedded block and sections assessed for the presence of intestinal metaplasia. TFF3 immunohistochemistry has consistently been shown to be a valuable adjunct that increases the accuracy of the CytospongeTM test by highlighting early goblet cells which may be missed on morphological assessment and by allowing pseudogoblet cells to be differentiated from true goblet cells.

Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry (Erratum).

The erratum corrects an error in the article "Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry."

Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry.

Photoacoustic tomography (PAT) is intrinsically sensitive to blood oxygen saturation (sO2) in vivo. However, making accurate sO2 measurements without knowledge of tissue- and instrumentation-related correction factors is extremely challenging. We have developed a low-cost flow phantom to facilitate validation of PAT systems. The phantom is composed of a flow circuit of tubing partially embedded within a tissue-mimicking material, with independent sensors providing online monitoring of the optical absorption spectrum and partial pressure of oxygen in the tube. We first test the flow phantom using two small molecule dyes that are frequently used for photoacoustic imaging: methylene blue and indocyanine green. We then demonstrate the potential of the phantom for evaluating sO2 using chemical oxygenation and deoxygenation of blood in the circuit. Using this dynamic assessment of the photoacoustic sO2 measurement in phantoms in relation to a ground truth, we explore the influence of multispectral processing and spectral coloring on accurate assessment of sO2. Future studies could exploit this low-cost dynamic flow phantom to validate fluence correction algorithms and explore additional blood parameters such as pH and also absorptive and other properties of different fluids.

Magnetic Resonance Imaging Is More Sensitive Than PET for Detecting Treatment-Induced Cell Death-Dependent Changes in Glycolysis.

Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death.

Oxygen-Enhanced and Dynamic Contrast-Enhanced Optoacoustic Tomography Provide Surrogate Biomarkers of Tumor Vascular Function, Hypoxia, and Necrosis.

Measuring the functional status of tumor vasculature, including blood flow fluctuations and changes in oxygenation, is important in cancer staging and therapy monitoring. Current clinically approved imaging modalities suffer long procedure times and limited spatiotemporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that may overcome these challenges. By acquiring data at multiple wavelengths, OT can interrogate hemoglobin concentration and oxygenation directly and resolve contributions from injected contrast agents. In this study, we tested whether two dynamic OT techniques, oxygen-enhanced (OE) and dynamic contrast-enhanced (DCE)-OT, could provide surrogate biomarkers of tumor vascular function, hypoxia, and necrosis. We found that vascular maturity led to changes in vascular function that affected tumor perfusion, modulating the DCE-OT signal. Perfusion in turn regulated oxygen availability, driving the OE-OT signal. In particular, we demonstrate for the first time a strong per-tumor and spatial correlation between imaging biomarkers derived from these in vivo techniques and tumor hypoxia quantified ex vivo Our findings indicate that OT may offer a significant advantage for localized imaging of tumor response to vascular-targeted therapies when compared with existing clinical DCE methods.Significance: Imaging biomarkers derived from optoacoustic tomography can be used as surrogate measures of tumor perfusion and hypoxia, potentially yielding rapid, multiparametric, and noninvasive cancer staging and therapeutic response monitoring in the clinic.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5980/F1.large.jpg Cancer Res; 78(20); 5980-91. ©2018 AACR.