Imaging extracellular vesicles arising from apoptotic tumour cells for cancer diagnosis and monitoring

As a large part of all health-related research is focused on cancer, and with several diagnostic and therapeutic procedures continuously emerging, the fact that this disease remains mostly uncured often seems overwhelming. Cancer is a disease with extremely heterogenous causes and biologic backgrounds, and multiple mechanisms have been identified as cancer-promoting, acting on several stages of the tumour progression. Among numerous other networks, cancer cells use their own death in order to signal an urgency for survival to their neighbouring cells. It has been observed that while a cancer cell is undergoing apoptosis, it can release signals which upon receipt by surrounding cells can promote the growth of tumour. Apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data indicate that, besides its role in tumour suppression, it can also promote oncogenic proliferation. Highly aggressive tumours such as Burkitt Lymphoma (BL) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. The network of regeneration and tissue repair mechanisms driven by cell-death has been named as the "onco-regenerative niche" by our group, and it is hypothesized that amongst other elements, extracellular vesicles (EVs) are key mediators of apoptotic cell-derived tumour microenvironment signals. EVs are membrane delimited structures secreted by cells, containing multiple types of bioactive material, including markers of the tissue they originate from. They are released by almost all cells and during several phases of the cell life cycle. EVs show numerous applications in diagnostics, and there is an increasing interest in their biological functions. However, mainly because of their small size and heterogeneity, there are challenges associated with their analysis, and although EVs are gaining popularity in clinical diagnostic practice, the guidelines for analytic procedures have not been established to date. Because the vesicles are much smaller than cells and fall in the category of nanoparticles, the methods which can be applied for their analysis are dedicated to smaller entities or are special adaptations of other methods routinely used for larger particles such as cells. Here, we report on EVs released by apoptotic BL cells (Apo-EVs) in relation to their potential use as cancer biomarkers in lymphoma. The hypothesis of this project examines the Apo-EVs as to their distinct structural and biochemical characteristics which can be used in the context of disease diagnosis and monitoring. As Apo-EVs can reach the main blood circulation, the analysis of Apo-EVs in patients can provide with information about the stages and the progression of the disease. The two main axes this work move around on are firstly, the structural and biochemical analysis of the Apo-EVs in order to examine their special molecular characteristics which render those different from other EVs which are not related to apoptosis and secondly, the study of how Apo-EVs interact with cells present in the blood and whether their cargo can be transferred to the second. Those two sets of studies can provide a better understanding of Apo-EVs and their roles, aiming at contributing towards the development of a disease monitoring platform. This project is focused on analytical platforms and techniques which can be applied to the nano-scale for imaging EVs in pre-clinical research and with the potential for application on patient samples. In particular, EVs released in vitro by Burkitt Lymphoma cells undergoing apoptosis upon UV irradiation are used throughout this study. Basic physical properties of Apo-EVs such as structure, size distribution, surface charge and membrane fluidity are discussed using Cryo Electron Microscopy (EM) and tomography, Nanoparticle Tracking Analysis, Dynamic Light Scattering and fluorescence anisotropy respectively. For phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission EM, fluorescence microscopy and quantitative PCR. The second part of the analysis consists of a study of the interaction of Apo-EVs with blood components such as platelets, leukocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. For this purpose, cells and platelets from human blood were co-incubated with Apo-EVs in order to examine the uptake and the possibility of Apo-EV cargo delivery intracellularly. Looking at the differences between Apo- and non-Apo-EVs, the Apo-EVs have a larger diameter, while structurally, the two populations are not different. However, we have identified distinct Apo-EV markers such as active caspase 3 and histone-3, or DNA and small non-coding RNA-Y. There is also a strong interaction of EVs with platelets and leukocytes but not with red cells, indicating potential mechanisms of transfer of EV cargo in the circulation. It was also found that this interaction does not only concern the surface of the cells, but EVs can enter the platelets or cells, which supports the hypothesis that their special biochemical cargo can be transferred inside those cells. It is concluded that for the characterization of the heterogenous Apo-EV populations, comparison of results from of each method is essential for choosing the appropriate combination of analytical tools. Finally, we consider that the monitoring free circulating Apo-EV or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response.