Characterising Mer and Axl receptor tyrosine kinase expression and transcriptomic profiling of myeloid cells in hepatocellular carcinoma

Abstract

Background: TAM receptor tyrosine kinases attenuate pro-inflammatory signalling in myeloid cells and maintain tissue homeostasis through apoptotic cell clearance (efferocytosis). Two members of the TAM-RTK family, MerTK and Axl, are overexpressed in human cancers; MerTK+ macrophages display a regulatory phenotype (M2c) similar to tumour associated macrophages (TAMs). MerTK+ and Axl+ macrophages contribute to immune paresis in liver disease. Little is known about MerTK and Axl expressing myeloid cells in HCC. In this thesis I characterise their expression and phenotype in circulating and tissue-resident myeloid cells, explore drivers for their expansion and function in vitro and utilise transcriptomics to interrogate wider TAM phenotype in human HCC.

Methods: Tissue and blood were collected from patients undergoing surgery or awaiting treatment for HCC. TAM-RTK expressing myeloid cells were identified using immunohistochemistry. Flow cytometry of isolated immune cell populations was utilised to understand their abundance and phenotype; in vitro conditioning and co-culture experiments were devised to recapitulate the tumour microenvironment, identify potential drivers for MerTK and Axl expression and assay their function. Serum and tissue homogenates were analysed for levels of TAM-RTK ligands. Transcriptomic analysis of tumour and liver derived myeloid cells was undertaken.

Results: MerTK+ macrophages are evident within inflammatory infiltrates in HCC. There is modest expansion of myeloid cells expressing MerTK and Axl in the tumour microenvironment, however gene expression is not upregulated and neither are downstream signalling cascades. In vitro conditioning does stimulate Axl but not MerTK expression and promotes immune-regulatory cytokine production. TAMs exhibit a ‘post-phagocytic’ phenotype with upregulation of C1Q, scavenger receptor Stabilin-1 and APOE.

Conclusions: TAM-RTK signalling is not activated within the tumour microenvironment. Transcriptomic analysis has identified an immune regulatory post-phagocytic and efferocytotic phenotype; further work is needed to evaluate the significance of this in the tumour biology of HCC, if it is not mediated through MerTK and Axl signalling.