Colorectal cancer metastasizes predominantly to the liver but also to the lungs and the peritoneum. The presence of extra-hepatic metastases limits curative (surgical) treatment options and is associated with very poor survival. The mechanisms governing multi-organ metastasis formation are incompletely understood. Here, we tested the hypothesis that the site of tumor growth influences extra-hepatic metastasis formation. To this end, we implanted murine colon cancer organoids into the primary tumor site (i.e., the caecum) and into the primary metastasis site (i.e., the liver) in immunocompetent mice. The organoid-initiated liver tumors were significantly more efficient in seeding distant metastases compared to tumors of the same origin growing in the caecum (intra-hepatic: 51 vs. 40%, p = 0.001; peritoneal cavity: 51% vs. 33%, p = 0.001; lungs: 30% vs. 7%, p = 0.017). The enhanced metastatic capacity of the liver tumors was associated with the formation of ‘hotspots’ of vitronectin-positive blood vessels surrounded by macrophages. RNA sequencing analysis of clinical samples showed a high expression of vitronectin in liver metastases, along with signatures reflecting hypoxia, angiogenesis, coagulation, and macrophages. We conclude that ‘onward spread’ from liver metastases is facilitated by liver-specific microenvironmental signals that cause the formation of macrophage-associated vascular hotspots. The therapeutic targeting of these signals may help to contain the disease within the liver and prevent onward spread.

Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85–0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases.