'UNMET' - Uncovering Mechanisms and Establishing Strategies to Target Vessel Co-Opted Colorectal Cancer Liver Metastases

Project Information

UNMET

Grant agreement ID: 101155460

DOI

10.3030/101155460

Project closed

EC signature date 24 April 2024

Start date 1 June 2024

End date 31 May 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 224 702,40

Coordinated by

AARHUS UNIVERSITET
Denmark

Periodic Reporting for period 1 - UNMET ('UNMET' - Uncovering Mechanisms and Establishing Strategies to Target Vessel Co-Opted Colorectal Cancer Liver Metastases)

Reporting period: 2024-06-01 to 2026-05-31

Summary of the context and overall objectives of the project

Colorectal cancer is one of the leading causes of cancer-related death worldwide, and the liver is its most common site of metastasis. The growth of colorectal cancer liver metastases depends on access to a blood supply. While some metastases stimulate the formation of new blood vessels, others use the liver's pre-existing vasculature through a process known as vessel co-option. This alternative vascularization strategy is associated with resistance to anti-angiogenic therapies, which are designed to inhibit new blood vessel formation.
The UNMET project aimed to improve our understanding of the mechanisms underlying vessel co-option in colorectal cancer liver metastases and to identify biological features that could support future therapeutic and diagnostic strategies. The project focused particularly on interactions between cancer cells and endothelial cells, which form the inner lining of blood vessels, and on understanding how these cells adapt to the metastatic tumour microenvironment.
To address these questions, UNMET combined the analysis of human colorectal cancer liver metastases with advanced single-cell and single-nucleus sequencing technologies and patient-derived three-dimensional tumour models. By comparing metastases with different growth patterns, the project aimed to identify cellular and molecular mechanisms associated with vessel co-option and provide a basis for developing new strategies for patients whose tumours are poorly responsive to existing anti-angiogenic treatments.

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

UNMET analysed human colorectal cancer liver metastases representing different histopathological growth patterns, together with adjacent liver tissue, using single-nucleus RNA sequencing. During the project, existing methods for isolating nuclei from frozen tumour tissue were found to provide suboptimal recovery of several biologically important cell populations including endothelial and tumor cells. The workflow was therefore systematically optimized, resulting in improved recovery of intact nuclei and increased representation of tumour and endothelial cells.
In parallel, a three-dimensional experimental model combining patient-derived colorectal cancer organoids with human liver sinusoidal endothelial cells was established. A workflow was developed to recover viable single cells from these complex co-cultures for single-cell RNA sequencing, enabling tumour–endothelial interactions to be investigated at high molecular resolution.
These approaches enabled detailed characterization of endothelial heterogeneity in colorectal cancer liver metastases. Distinct endothelial states were identified across adjacent liver and metastatic tissue, including vascular changes associated with vessel co-option and adaptation to the tumour microenvironment. The results highlighted endothelial mechanosensing and mechanoadaptation as prominent features of replacement-type metastases. Experimental characterization of the physical tumour microenvironment, including atomic force microscopy-based analyses, was initiated and remained ongoing at the end of the project.
The project also contributed to a collaborative investigation of the immune microenvironment of colorectal cancer liver metastases. This work identified a spatially organized immunosuppressive niche enriched in SPP1-expressing macrophages in replacement-type metastases.
UNMET established optimized experimental and sequencing workflows, generated a human single-nucleus transcriptomic dataset of colorectal cancer liver metastases, developed a sequencing-compatible tumour–endothelial co-culture model, and identified endothelial mechanoadaptation and immune microenvironment features associated with vessel co-option.

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)

UNMET generated methodological and biological advances beyond the current state of the art in the study of vessel co-option in colorectal cancer liver metastases. A major methodological result was the development of an optimized nuclei isolation workflow for snap-frozen human metastatic tissue. The approach improves the recovery of biologically relevant cell populations, particularly tumour and endothelial nuclei, which are often underrepresented in single-nucleus transcriptomic studies of complex liver tumours. In parallel, a sequencing-compatible three-dimensional co-culture model combining patient-derived colorectal cancer organoids and human liver sinusoidal endothelial cells was established, providing a platform to investigate tumour–vascular interactions at single-cell resolution.
At the biological level, the project identified marked endothelial heterogeneity in human colorectal cancer liver metastases and highlighted mechanosensing and mechanoadaptation as prominent features associated with vessel co-option. These findings extend the conventional view of vessel co-option as primarily a tumour-cell-driven process by indicating that endothelial adaptation to the physical and cellular tumour microenvironment may also contribute to the maintenance of co-opted vessels. The project therefore provides new candidate mechanisms for understanding resistance to therapies targeting angiogenesis.
In addition, collaborative analyses identified a spatially organized SPP1-expressing macrophage niche associated with replacement-type metastases, further demonstrating that vessel-co-opted tumours are characterized by coordinated vascular and immune microenvironmental changes.
The results provide new experimental tools, human molecular datasets and candidate mechanisms for future studies of vessel co-option. Further functional validation, including completion of tissue-mechanics analyses and in vivo investigation of prioritized targets, will be required to establish therapeutic relevance. Future studies should also assess whether the identified endothelial and immune signatures can support patient stratification or prediction of response to anti-angiogenic therapy.