Restoration of paracrine signalling within the tumour microenvironment increases tumour growth and activation of c-Met


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Richard Argent1, Rajendra Kumari1, Phil Clarke1, David Onion1, Dileep Lobo2, Anna Grabowska1, Peter King3, Brett Hall3, Martin Page3, Sue Watson1
1Ex Vivo Cancer Pharmacology Centre Of Excellence, Division Of Pre-Clinical Oncology, University Of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom,2Division Of Gastrointestinal Surgery, Nottingham Digestive Diseases, Biomedical Research Unit, Nottingham University Hospitals, Queen's Medical Centre, Nottingham, United Kingdom,3OrthoBiotech, Oncology Research & Development, Beerse, Belgium

Background

Paracrine signalling between tumour epithelial cells and the stroma in the tumour microenvironment is a pre-requisite for tumour growth and may determine responsiveness to therapeutic inhibition. Hepatocyte growth factor (HGF) and the HGF receptor, c-Met, are reported to be up-regulated in colorectal liver metastases (CRLMs) and considered to be markers of metastatic potential. They also promote epithelial-mesenchymal transition (EMT), an important developmental process which is frequently activated in carcinogenesis resulting in metastatic spread.

Method

CRLMs (n=27) were grown as sub-cutaneous xenografts in nude mice, and subsequently treated with c-Met inhibitors. Tissues were used for RNA extraction and quantitative PCR analysis, were enzymatically disaggregated to isolate epithelial and mesenchymal cells, and were used for immunohistochemistry.

Results

Analysis of primary human CRLMs showed that c-Met was overexpressed and accompanied by decreased E-cadherin, and increased EMT-related gene expression. Enrichment of individual cell types from primary CRLMs showed that HGF was mesenchymal, whereas c-Met was epithelial. Growth of tumours as sub-cutaneous xenografts revealed that human stroma, and thus paracrine signalling, was rapidly lost, and treatment of tumour-bearing mice with c-Met inhibitors had a generally poor response. Co-implantation of primary CRLM tumour epithelial cells with tumour-conditioned mesenchymal cells increased tumour growth, decreased necrosis, and increased c-Met activation. Implantation of tumour-associated fibroblasts into xenografts was also found to increase response to a c-Met inhibitor and to standard of care agents.

Conclusion

Paracrine HGF:c-Met signalling is rapidly lost in CRLM xenograft models indicating the need to restore human stroma, and it's loss may reflect the poor response to inhibition. Recapitulation of the human tumour microenvironment in xenografts by implantation of human mesenchymal cells improves the therapeutic response, and therefore provide improved models to assess drug efficacy.