The expression of CD73 in hepatocellular carcinoma and its microenvironment in the context of chronic inflammation

Faulkes, Rosemary Elizabeth (2022). The expression of CD73 in hepatocellular carcinoma and its microenvironment in the context of chronic inflammation. University of Birmingham. M.Sc.

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Abstract

Introduction
Primary liver cancer, hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. HCC often develops in the context of liver fibrosis which contributes to an immunosuppressive tumour microenvironment (TME). Overcoming the TME in HCC is a major challenge to successful therapy. Better understanding of the cell specific contribution to immunosuppression in the TME is required to help boost current immunotherapy. Hepatic sinusoidal endothelial cells (HSEC) are the gatekeeper for immune cell recruitment and pilot RNA sequencing data showed significant upregulation of CD73 in tumour endothelium. CD73 exerts an immunosuppressive effect through production of adenosine in the extracellular space. We sought to understand its expression in HCC and potential role in HSEC.

Methods
Immunohistochemistry for CD73 was performed on both normal liver (NL) and a range of chronic liver diseases (CLD) as well as a cohort of 99 HCC samples. Qualitative and quantitative analysis was performed and results compared with clinical data for the HCC cohort. Quantitative RT-PCR for canonical CD73 and a spliced variant was undertaken on whole liver tissue and hepatocyte cell lines Huh7 and HepG2. Primary HSEC isolated from liver tissue using a magnetic bead technique were analysed for CD73 expression by immunofluorescence and RT-PCR. CD73 expression was also studied in HSEC in static conditions and compared to HSEC that had been subject to shear stress.

Results
Immunohistochemical staining was consistent with CD73 expression on endothelial and epithelial cells in normal liver and CLD. Staining of the canaliculi was noted to be more pronounced in biliary disease compared to NL. The expression pattern of CD73 in HCC was heterogeneous, with a variation between membranous and cytoplasmic expression on tumour hepatocytes. Membranous expression was associated with a trend towards increased incidence of vascular invasion, a marker of poor prognosis, and worse overall survival, although this did not reach statistical significance. In contrast, peri-tumour vasculature was positive for CD73 in all cases of HCC in the cohort.
At the transcript level, the spliced variant of CD73 (CD73S) was minimally expressed in NL tissue, but increased in CLD tissue, particularly biliary disease.
Isolated HSEC maintained expression of CD73 in culture and gene expression was detected in both HSEC and the hepatocyte cell lines, Hep G2 and Huh-7. The spliced variant of CD73 was significantly higher in Huh-7 than HSEC. There was no difference in gene expression due to shear stress.

Conclusion
I found that CD73 is expressed on the sinusoids and canalicular structures of both normal liver and chronic inflammatory disease. CD73 expression was variable in HCC, with membranous and cytoplasmic staining in tumour cells. In all cases of HCC, the peritumoural vasculature was positive for CD73, which may explain the upregulation of CD73 in previous RNAseq studies of tumour versus non-tumour endothelium. HSEC maintain their expression of CD73 in culture. The CD73 spliced variant is significantly upregulated in the hepatoma cell line Huh 7 at the transcript level, and may reflect the cytoplasmic staining of CD73 on IHC that was unique to HCC tumours. This work highlights the need for further studies into the function of CD73 in HSEC especially around immune cell activation and recruitment to the TME of HCC and its future potential as a vascular therapeutic target.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):