Investigating the role of primary cilia loss in intrahepatic cholangiocarcinoma

Abstract

Primary cilia (PC) are important sensory organelles which protrude from the epithelial cells lining bile ducts (cholangiocytes), where they serve as sensors of bile composition, flow and osmolality. PC malformation has been reported in cholangiocarcinoma (CCA), a neoplasia that originates from these biliary epithelial cells. Despite changes in cilia regulation within CCA, PC-loss alone is insufficient for CCA initiation. However, we show that when PC is deleted from biliary cells in an intrahepatic CCA (iCCA) mouse model, tumour burden significantly increases. Interestingly, these non-ciliated tumour cells do not show significant differences in cell proliferation when compared to ciliated cells. In this thesis, I explore the transcriptional differences between ciliated and non-ciliated iCCA tumour cells and ask how these changes influence the immune cells that surround emerging tumours.

Using an iCCA mouse model in which Trp53 and Pten are deleted alongside the essential cilia gene Wdr35, I found that loss of cilia accelerated tumour development. In the absence of chronic inflammation, cilia loss in the context of tumour suppressor loss accelerated the development of biliary neoplasia. Bulk RNA sequencing of these tumour cells demonstrated that PC-loss increased the expression of pro-inflammatory cytokines such as Cxcl9, Cxcl10 and Cxcl11. However, the expression of neutrophil-specific chemokines such as Cxcl1, Cxcl2 and Cxcl5 was significantly suppressed. In concordance, immunohistochemical analysis revealed that neutrophil infiltration was frequently decreased in the livers of mice with non-ciliated biliary cells when compared to mice with ciliated cholangiocytes. To further explore tumour-neutrophil interactions in vitro, I derived organoid lines from biliary cells isolated from ciliated and non-ciliated mouse models of iCCA and showed that the expression and secretion of neutrophil-specific chemokines was significantly downregulated in non-ciliated organoids. I then isolated naïve WT bone marrow neutrophils and co-cultured these with organoid conditioned media (CM). In a Transwell migration assay, I showed that neutrophils exposed to non-ciliated CM migrated significantly less than when cultured in the presence of ciliated CM, suggesting that PC-loss on cancer cells directly impacts neutrophil migration.

The work presented here demonstrates how losing primary cilia can promote a more immunosuppressive microenvironment during iCCA development and highlights the importance of continuing to address the status and role of primary cilia in future iCCA studies.