The facilitative role of the extracellular matrix in the development of colorectal cancer liver metastases

We have investigated the hypothesis that a desmoplastic reaction (DR), characterised by the deposition of collagens I and III produced by activated stromal cells offers Colorectal Cancer (CRC) liver metastases a growth and survival advantage. Immunohistochemical staining of liver specimens obtained at resection for CRC, demonstrated increased deposition of fibrillar collagens and alterations in collagen IV distribution within the tumour as part of a DR. In addition the deposited fibrillar collagens were closely associated with increased numbers of activated hepatic stellate cells/myofibroblasts. β1 integrins were highly expressed by both cancer & stromal cells throughout the tumour stroma. However, in poorly differentiated areas of the CRC metastases β1 integrins appeared to be down-regulated, with αv integrin (especially αvβ5) expression upregulated. Collagen I used as a tissue culture substrate significantly enhanced the growth of the CRC cell lines (HT-29 & KM12 cell lines series) compared to both control (plastic) and collagen IV (normal basement membrane component). Clonogenic (survival) and PARP cleavage (apoptosis) assays, showed that collagen I compared to collagen IV significantly increased the survival and reduced the rate of cellular apoptosis for CRC treated with chemotherapy (5-Fluorouracil). The adhesion and proliferation of CRC cells on collagens I and IV was significantly reduced in a dose dependent manner, after incubation with β1 integrin neutralizing antibodies (5-10μg/ml), compared to IgG controls. In contrast αvβ3 and αvβ5 neutralizing antibodies (5-20μg/ml), had no influence on the CRC cell adhesion, but significantly reduced the rate of proliferation of the CRC cell lines on collagens; especially for the highly metastatic KM12SM cell line. By demonstrating a reduction in proliferation in response to MMP resistant r/r collagen we have further reinforced our αv integrin neutralising antibody experiments, complementing them with a model using a dominant negative ligand. These results support a role for the desmoplastic reaction in supporting CRC metastases, mediated via β1 and αv integrins. As CRC adopt a more aggressive malignant phenotype, matrix turnover reveals specific binding epitopes which upon ligation by αv integrins plays a key growth regulatory role.