Recirculated tissue macrophages (TiMa) in blood: Novel approach to early diagnosis and treatment monitoring in oncology

Project Information

TiMaScan

Grant agreement ID: 695655

DOI

10.3030/695655

Project closed

EC signature date 21 October 2016

Start date 1 November 2016

End date 31 October 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 500 000,00

EU contribution

€ 2 500 000,00

Coordinated by

ACADEMISCH ZIEKENHUIS LEIDEN
Netherlands

Periodic Reporting for period 5 - TiMaScan (Recirculated tissue macrophages (TiMa) in blood: Novel approach to early diagnosis and treatment monitoring in oncology)

Reporting period: 2022-11-01 to 2023-10-31

Summary of the context and overall objectives of the project

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 to improve cure rates and increase quality-of-life. However, many 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 actively recruited to tissue damage, including tumor sites where they phagocytose apoptotic tumor cells. Once they have fulfilled their local tissue-cleaning task, they 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.
This project aimed 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) is being developed into a novel tool for early diagnosis and treatment monitoring in oncology, focusing on colon, lung, breast, prostate cancer and melanoma. TiMaScan diagnostics should be minimally-invasive (~2ml of blood), rapid, accurate, broadly available and cost-effective, only requiring a flow cytometer and appropriate antibodies against tissue-specific and/or cancer-related protein fragments. TiMaScan diagnostics might also be applicable for early diagnosis and disease monitoring in other medical conditions, in which tissue damage and/or inflammation plays a role (Figure 1).

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The TiMaScan project initially focused on characterization of the multiple subpopulations of monocytes, macrophages and dendritic cells in different tissues (e.g. bone marrow, peripheral blood, peritoneal dialysis samples, skin, and colon). 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 the identification of new (minor) subsets in peripheral blood (Figure 2). 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). This novel information allowed for optimized 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, has been applied to profile the heterogeneous composition of colorectal cancers. The related data search has identified four 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. To understand how phagocytic cells break down (digest) cancer cells into fragments, we developed a new tool for isolation of subcellular compartments (lysosomes and phagolysosomes), where digestion takes place. This allowed for identification of the proteases involved in the digestion process to understand the digestion patterns in monocytes, macrophages and dendritic cells and to reveal the cancer-derived protein fragments which can potentially be used for early diagnosis and treatment monitoring in oncology.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Thanks 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, allowed the design of a single-tube flow cytometry panel for the identification of >25 innate myeloid cells in peripheral blood and > 35 in bone marrow. This tube can be employed for immune-monitoring in multiple clinical/diagnostic settings (Figure 3). This resulted in the PCT/NL2020/050688 patent application (priority date: 5 November 2020).
Whereas conventional proteomics uses millions of cells, we wished to assess the best strategy to analyze very low numbers of cells in order to characterize the proteome of the different monocyte, macrophage and dendritic subsets. Based on comparative studies and novel designs, a micro-method was developed, which allows for identification of up to 900 proteins from 2,500 cells, only. This allowed in-depth proteome investigation of many innate cell subsets.
So far, limited information has been available on the phagolysosomal digestion processes and the resulting peptides. The combined information from the above studies provided new insight into the complexity of intra-phagolysosomal digestion processes (“digestomcs”). Dependent on differences in protease contents of the lysosomes between different innate cells (monocytes, macrophages and dendritic cells), the fragments of the same protein might differ.
This protease information was included in special software to predict how different proteins might be digested into peptide fragments and explains why the vast majority of classical antibodies cannot recognize anymore tissue-specific protein fragments in phagolysosomes of macrophages. This also explains why pathologists can identify tissue-specific and tumor-specific proteins in local tissue macrophages (at the tumor site), while such signals are lost in distant draining lymph nodes.
This novel understanding of “digestomics” will significantly progress the TiMaScan research program. In fact, the majority of (commercially) available antibodies cannot be used for the TiMaScan project. Instead, completely new antibodies have to be designed against well-defined protease-mediated peptides, derived from the tissue-specific and/or tumor-related proteins.
If indeed such new antibodies can be made available, the TiMaScan concept will be applicable in a clinical setting, as a minimally invasive method for cancer screening and monitoring. Such method has the potential for being 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.

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