Origin and function of the Stroma in cholangiocarcinoma.
Robson, Andrew John
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Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.