Transglutaminase-2 (TG2) is the most highly and ubiquitously expressed member of the transglutaminase enzyme family and is primarily involved in protein cross-linking. TG2 has been implicated in the development and progression of numerous cancers, with a direct role in multiple cellular processes and pathways linked to apoptosis, chemoresistance, epithelial-mesenchymal transition, and stem cell phenotype. The tumour microenvironment (TME) is critical in the formation, progression, and eventual metastasis of cancer, and increasing evidence points to a role for TG2 in matrix remodelling, modulation of biomechanical properties, cell adhesion, motility, and invasion. There is growing interest in targeting the TME therapeutically in response to advances in the understanding of its critical role in disease progression, and a number of approaches targeting biophysical properties and biomechanical signalling are beginning to show clinical promise. In this review we aim to highlight the wide array of processes in which TG2 influences the TME, focussing on its potential role in the dynamic tissue remodelling and biomechanical events increasingly linked to invasive and aggressive behaviour. Drug development efforts have yielded a range of TG2 inhibitors, and ongoing clinical trials may inform strategies for targeting the biomolecular and biomechanical function of TG2 in the TME.

Colorectal cancer is the third most common cancer worldwide, and the fourth leading cause of malignancy-related mortality. This highlights the need to understand the processes driving this disease in order to develop new treatments and improve patient outcomes. A potential therapeutic target is the increased stiffness of the tumour microenvironment, which is linked to aggressive cancer cell behaviour by enhancing biomechanical signalling. In this study, we used an siRNA-based approach to investigate the contribution of the protein cross-linking enzyme transglutaminase-2 (TG2) to matrix remodelling and biomechanical properties of the tumour microenvironment. TG2 inhibited cancer cell growth in organotypic 3D fibroblast/SW480 co-culture models, and biomechanical analysis demonstrated that colorectal cancer cells induced fibroblast-mediated stiffness which was inhibited by silencing TG2. These biomechanical changes were associated with observed alterations to collagen fibre structure, notably fibre thickness. Our in vitro findings of collagen composition changes were also seen with imaging biopsied tissues from patients with colorectal cancer, with TG2 correlating positively with thicker collagen fibres, and associating with poor outcome as determined by disease recurrence post-surgery and overall survival. In conclusion, this study demonstrates a role for TG2 in the stromal response to invading tumour, leading to tissue stiffening and poor outcome in patients.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as ‘C5’) photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Background/Objectives: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with poor outcomes in advanced stages and significant limitations in current chemotherapy regimens due to systemic toxicity. Extracellular vesicles (EVs) have emerged as promising natural drug delivery vehicles, offering the potential for targeted, less toxic therapies. This study investigates the feasibility of using autologous, patient-derived EVs as a delivery system for the chemotherapeutic agent doxorubicin, focusing on how disease stage and the EV protein corona influence loading and delivery efficiency. Methods: EVs were isolated from plasma and tissue samples of CRC patients at different disease stages, as well as from healthy controls, demonstrating successful isolation and characterisation of EVs, with distinct profiles across different sources. Results: Doxorubicin loading into EVs was significantly higher in CRC patient-derived EVs compared to healthy controls, and tissue-derived EVs yielded higher quantities of drug-loaded particles. Delivery of doxorubicin-loaded EVs to recipient CRC cell lines (SW480 and SW620) revealed that disease stage impacts both EV uptake and drug delivery, with late-stage EVs showing reduced uptake and delivery efficiency. The protein corona, known to coat circulating EVs, was found to influence drug loading and delivery. Pre-treatment of cell line-derived EVs with plasma proteins enhanced EV uptake but reduced doxorubicin loading and subsequent delivery, particularly when using plasma from healthy volunteers. Conclusions: These findings underscore the importance of EV source and protein corona composition in optimising drug delivery strategies. Our results suggest that autologous, patient-derived EVs hold potential as a targeted drug delivery system for CRC, but highlight the need for further optimisation of EV isolation, loading methods, and understanding of how disease progression affects EV functionality. This approach could ultimately reduce systemic toxicity and improve therapeutic outcomes for CRC patients.

Malignant mesothelioma (MM) is a rare cancer caused by exposure to asbestos, this condition continues to represent a significant diagnostic and therapeutic challenge. Due to the long disease latency, non-invasive diagnostic modalities such as breath analysis may enhance MM early detection by identifying the disease specific pattern of volatile organic compounds (VOCs) in exhaled breath. In this study, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was used to extract VOCs from the headspace of two mesothelioma cell lines: MSTO-211H (biphasic mesothelioma) and NCI-H28 (epithelioid mesothelioma) in addition to MET-5a (non-malignant mesothelial cell line), following exposure to asbestos mineral fibers (actinolite, amosite, crocidolite and chrysotile) and a non-asbestos fiber control (wollastonite). Multivariate statistical analysis was applied to identify VOC-based biomarkers associated with asbestos exposure within the cell lines. Data confirms that exposure to mineral fibers (including the non-asbestos fiber control) induces significant alterations in the levels of as many as 24 VOCs in each cell line. This is the first study to investigate the impact of asbestos mineral fiber exposure on the VOC profile of MM cell lines. This may be relevant to studies involving mesothelioma patients, as these VOCs could also serve as indicators of asbestos exposure in individuals who have been exposed to asbestos.