Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - TiMaScan (Recirculated tissue macrophages (TiMa) in blood: Novel approach to early diagnosis and treatment monitoring in oncology)
Teaser
In the ageing European population, cancer has become the most common cause of death. Consequently, there is a growing need for screening programs for early detection of cancer and methods for monitoring of treatment effectiveness (e.g. scanning/imaging technologies, serum...
Summary
In the ageing European population, cancer has become the most common cause of death. Consequently, there is a growing need for screening programs for early detection of cancer and methods for monitoring of treatment effectiveness (e.g. scanning/imaging technologies, serum assays and regular biopsies) to improve cure rates and increase quality-of-life. However, in many cases the screening and monitoring methods do not provide sufficient sensitivity and specificity and/or are highly invasive. Thus, novel diagnostic techniques are required.
Sensitive intra-tissue total body scanning is continuously performed by the monocyte/macrophage system. These cells are modulated by tumor-releasing factors and are actively recruited to the tumor sites where they engulf apoptotic/necrotic tumor cells. Once they have fulfilled their local tissue-cleaning task, they can migrate via lymph vessels to lymph nodes to present (tumor-) antigens to T-cells and potentially recirculate to the blood stream, where they can be monitored and evaluated for their phagolysosomal contents by flow cytometry.
Therefore, this project aims to unravel phagocytosis of cancer cells, their digestion into tissue-specific and/or cancer-related protein fragments, the migration/recirculation of tissue macrophages (TiMas) to blood, and the detection of intra-phagolysosomal protein fragments in blood TiMas by antibodies. Building on this information, flow cytometric scanning of blood TiMas (TiMaScan) will be developed into a novel tool for early diagnosis and treatment monitoring in oncology, focusing on colon, lung, breast and prostate cancer. TiMaScan diagnostics will be minimally-invasive (1-2ml of blood), rapid, accurate, broadly available and cost-effective, only requiring a flow cytometer. Also, it can be applicable for early diagnosis and disease monitoring in other medical conditions, in which tissue damage and/or inflammation plays a role.
Work performed
During the period covered by this report (November 1st 2016 to April 30th 2019), the TiMaScan research focused on characterization of the different subpopulations of monocytes, macrophages and dendritic cells (components of the immune system) in different tissues (e.g. bone marrow, peripheral blood, peritoneal dialysis samples, skin, normal colon and lymph nodes). In order to do so, flow cytometric strategies were applied to define the markers (and corresponding antibodies) required for the identification of these subsets. This resulted in identification of new, small cell populations in peripheral blood. The obtained information allowed the design of tools for studying these new subpopulations in multiple clinical settings and to further explore the relationship between the different populations in distinct tissues (e.g. using high-end flow cytometry and mass cytometry techniques). The information generated by these studies will improve the understanding of how the monocyte/macrophage system is regulated in health and disease. Also, the information obtained from the aforementioned extensive characterization allowed for the optimization of the methods for isolation of the same cells (cell sorting), to be used for additional investigations (transcriptomics, proteomics and morphology strategies). Because some cell populations of interest are present in very low numbers in tissues, an in-depth evaluation of five proteomics protocols for mass spectrometric analysis of their proteins was performed, from which a novel strategy based on usage of microbeads for protein purification showed the best results. This novel approach, in combination with transcriptomics studies, will be applied to profile the heterogeneous composition of colorectal cancers. A data search has already been started to identify the proteins specifically expressed in normal target tissues and/or related to cancer conditions.
Additionally, a new method was developed to evaluate the phagocytic ability (i.e. capacity of engulfment) of monocytes and dendritic cells. In line with this, and as we are interested in defining how phagocytic cells break down (digest) cancer cells into fragments, we are developing a new tool for isolation of subcellular compartments (lysosomes and phagolysosomes), where digestion takes place. This new method will allow for identification of the enzymes (proteases) involved in the digestion process to understand the digestion patterns of human monocytes, macrophages and dendritic cells and to reveal the cancer-derived protein fragments which could be potentially used for early diagnosis and treatment monitoring in oncology.
Final results
During the period comprised between November 1st 2016 to June 30th 2019, and in the context of the tasks performed in the TiMaScan project, several achievements have been accomplished. These activities mainly focused on fundamental research that aims at understanding the monocyte/macrophage system and on the development of new methods and strategies to be implemented in the next stages of the project.
In short, due to extensive characterization of monocytic cells in multiple tissues (over 100 proteins evaluated), new subpopulations of monocytes were identified in peripheral blood of adult individuals. These populations are functionally different and behave differently in case of disturbed homeostasis (e.g. tumors or damage induced by surgery). For evaluation of the phagocytic ability of these new subsets, a new flow cytometry-based method was developed, that can also be used for other applications. The results of the extensive characterization of monocytes and other innate myeloid cells, also allowed the design of a novel single-tube flow cytometry panel for the identification of >20 innate myeloid cells in peripheral blood, and which can be employed for immune-monitoring of these populations in a single step, also in other clinical/diagnostic settings.
Furthermore, in order to characterize the whole proteome of the different monocyte, macrophage and dendritic cell populations, evaluation of the best strategy to analyse very low numbers of cells was performed, and a micro-method for identification of up to 900 proteins from 2,500 cells was established. In this context it should be noted that conventional proteomics uses millions of cells.
Overall, these results highlight the development of knowledge and technology required to successfully fulfil the objectives proposed for the next period of the TiMaScan project. Completion of these objectives will allow for the identification of the protease repertoire in monocytes, macrophages and dendritic cells, and therefore to improve the understanding of the processes involved in phagolysosome digestion and antigen presentation, knowledge that has applications beyond the scope of the TiMaScan project (e.g. vaccination). In addition, the new knowledge on normal tissue and tumor cells, resulting from the extensive characterization of these cell (transcriptomics and proteomics of purified cells) will provide a better understanding of the selection processes and disease (cancer) progression. Furthermore, if the TiMaScan concept proves to be correct and applicable in a clinical setting, a flow cytometry-based, minimally invasive method for cancer screening and monitoring will be developed, which has the potential for being cheaper and faster than the currently used imaging techniques and being applicable to other fields where tissue damage plays a role, such as neurodegenerative diseases and insidious infections.