Maria A. Hawkins

Objective. Quantitative analysis of cellular morphology and spatial organisation within intact tissue remains challenging, particularly when three-dimensional information must be preserved. This study investigates whether laboratory propagation-based phase-contrast x-ray computed tomography (CT) can support nuclei-based quantitative analysis in unstained tissue while retaining the surrounding tissue architecture. Approach. We propose a nuclei-based quantitative imaging workflow that combines laboratory propagation-based phase-contrast x-ray CT with volumetric segmentation. Intact unstained liver tissue was imaged at cellular resolution, then nuclei were segmented throughout the reconstructed volume and quantitative metrics describing the nuclear morphology and spatial organisation were extracted. The resulting measurements were evaluated in two non-overlapping volumes of interest and compared with histological reference data using thickness-matched virtual CT slices. Main results. The proposed workflow enabled visualisation and segmentation of individual nuclei throughout intact unstained liver tissue. Comparable nuclear morphology and spatial organisation metrics were obtained from two non-overlapping volumes of interest, indicating stable segmentation performance within the analysed specimen. Comparison with H&E histology demonstrated broad agreement in nuclear morphology. Significance. This work demonstrates the feasibility of nuclei-based quantitative analysis using laboratory phase-contrast x-ray CT in intact unstained tissue. The approach provides volumetric cellular information while preserving tissue architecture and may complement conventional histological assessment in applications requiring three-dimensional analysis. These findings highlight the potential of laboratory phase-contrast CT for quantitative tissue characterisation and spatial cellular analysis.

SUMMARY: Rectal cancers are often treated with neoadjuvant chemoradiotherapy (CRT), yet 85% of patients do not achieve a pathological complete response. To identify the molecular determinants of CRT response, we profiled the single-cell signalling, DNA-damage, cell-cycle, apoptotic, and cell-fate responses of 2,769 patient-derived organoid cultures treated with CRT, cancer-associated fibroblasts (CAFs), and signal-rewiring agents. We find that CRT response is determined by stem cell-fate. CRT triggers comparable DNA-damage in isogenic proliferative (proCSC) and revival (revCSC) colonic stem cells, but proCSC retain damage and die whereas revCSC resolve damage and persist. Both CRT and CAFs drive proCSC to a common treatment-resistant revCSC fate and high revCSC predicts worse survival in patients. Pharmacologically constraining stem-cell plasticity increases CRT sensitivity, and Spatial Perturbation of ARrayed Tumour Assembloids (SPARTA) confirms YAP/TEAD inhibition improves chemotherapy responses in human stromal-tumour models. These results suggest that cancer cell-fate, not genotoxic damage itself, ultimately governs response to standard-of-care chemoradiotherapy. HIGHLIGHTS: Rectal cancer stem cell-fate determines chemoradiotherapy-induced apoptosis proCSCs retain DNA-damage and die, whereas revCSCs repair damage and persist CAFs and chemoradiotherapy converge on a common chemo-radioresistant revCSC state SPARTA reveals TEAD inhibition blocks DNA-repair persisters in stromal assembloids

Background: Oesophageal cancer is a major cause of morbidity and mortality. Around 40% of patients present with locally advanced disease. Outcomes are poor, with 5-year survival of around 50% for locally advanced oesophageal adenocarcinoma (OAC) and 60% for oesophageal squamous cell carcinoma (OSCC). Over a fifth of recurrences are locoregional. These may be reduced by the addition of chemoradiotherapy (CRT) to best pre-operative systemic anti-cancer therapy (SACT). However, photon-based CRT is associated with a higher frequency of complications than chemotherapy alone. This study will explore whether, in patients with locally advanced OAC and OSCC managed with hypofractionated CRT following best pre-operative systemic therapy, the use of proton beam therapy (PBT) reduces post-operative complications when compared with photon-based treatment. Methods/design: PROTIEUS is an investigator-initiated randomised multi-centre phase 2 trial aiming to recruit 170 patients with locally advanced OAC ( n = 130) or OSCC ( n = 40) from the UK National Health Service. Patients with locally advanced cT ≥ 2, N0-2 non-metastatic disease are eligible for inclusion and will be randomised 1:1 to receive 40.05 Gy (RBE) in 15 fractions over three weeks using either PBT or intensity modulated/rotational arc photon radiotherapy. All patients will receive three one-week cycles of concurrent intravenous carboplatin/paclitaxel. CRT will be delivered following best pre-operative SACT. The primary endpoint is the rate of severe post-operative complications within 90 days post-surgery (grade 3 or higher Clavien-Dindo classification and CTCAE v5.0). Secondary endpoints relate to efficacy (pathological complete response and clear resection margin rate, disease-free and overall survival), tolerability (completion of planned CRT regime, time to and completion of adjuvant therapy), morbidity (long-term quality of life measures) and health economics (cost-effectiveness analyses using EQ-5D-5 L and resource use assessments). Discussion: There is a need to improve local control in OAC and OSCC. Given only modest gains with perioperative immune checkpoint inhibition and a disease landscape in which there are few targets for precision or personalised therapies, there are limited options to achieve further gains across the disease population using SACT. This study will therefore determine which of PBT or photon-based pre-operative CRT is the most tolerable and least toxic for integration into existing systemic treatment paradigms. Trial registration: https://doi.org/10.1186/ISRCTN50098578 .