Current treatment for metastatic disease targets angiogenesis. With the increasing data demonstrating that cancer cells do not entirely rely on angiogenesis but hijack the existing vasculature through mechanisms such as co‐option of existing blood vessels, identification of targets has become of utmost importance. Our study looks at the vasculature of chemonaïve and treated colorectal carcinoma liver metastases (CRCLMs) to obtain a basic understanding of the microvessel density, type of vasculature (mature versus immature), and correlation with histopathological growth patterns that demonstrate unique patterns of angiogenesis. We performed immunohistochemistry on chemonaïve sections of desmoplastic histopathological growth pattern (DHGP) and replacement histopathological growth patterns (RHGP) lesions with CD31 [endothelial cell (EC) marker] and CD34/Ki67 double staining, which denotes proliferating ECs. The CD31 stains demonstrated a lower microvascular CD31 +ve capillary density in the DHGP versus RHGP lesions; and integrating both immunostains with CD34/Ki67 staining on serial sections revealed proliferating vessels in DHGP lesions and co‐option of mature existing blood vessels in RHGP lesions. Interestingly, upon treatment with chemotherapy and bevacizumab, the RHGP lesions showed no necrosis whereas the DHGP lesions had almost 100% necrosis of the cancer cells and in most cases there was a single layer of viable cancer cells, just under or within the desmoplastic ring. The survival of these cells may be directly related to spatial location and possibly a different microenvironment, which may involve adhesion to different extracellular matrix components and/or different oxygen/nutrient availability. This remains to be elucidated. We provide evidence that DHGP CRCLMs obtain their blood supply via sprouting angiogenesis whereas RHGP lesions obtain their blood supply via co‐option of existing vasculature. Furthermore current treatment regimens do not affect RHGP lesions and although they kill the majority of the cancer cells in DHGP lesions, there are cells surviving within or adjacent to the desmoplastic ring which could potentially give rise to a growing lesion.

Colorectal cancer liver metastasis (CRCLM) has two major histopathological growth patterns: angiogenic desmoplastic and non-angiogenic replacement. The replacement lesions obtain their blood supply through vessel co-option, wherein the cancer cells hijack pre-existing blood vessels of the surrounding liver tissue. Consequentially, anti-angiogenic therapies are less efficacious in CRCLM patients with replacement lesions. However, the mechanisms which drive vessel co-option in the replacement lesions are unknown. Here, we show that Runt Related Transcription Factor-1 (RUNX1) overexpression in the cancer cells of the replacement lesions drives cancer cell motility via ARP2/3 to achieve vessel co-option. Furthermore, overexpression of RUNX1 in the cancer cells is mediated by Transforming Growth Factor Beta-1 (TGFβ1) and thrombospondin 1 (TSP1). Importantly, RUNX1 knockdown impaired the metastatic capability of colorectal cancer cells in vivo and induced the development of angiogenic lesions in liver. Our results confirm that RUNX1 may be a potential target to overcome vessel co-option in CRCLM.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases.