Investigating the Intrinsic Role of Programmed Death-Ligand 1 in Human Cancers.

Programmed death-ligand 1 (PD-L1) expression is a survival mechanism employed by tumours to mediate immune evasion and tumour progression. PD-1/PD-L1-targeted therapies have revolutionised the cancer therapy landscape due to their ability to promote durable anti-tumour immune responses in select patients with advanced cancers. However, some patients are unresponsive, hyperprogressive or develop resistance. The exact mechanisms for this are still unclear. Recently, a pro-tumorigenic role of PD-L1 to send pro-survival signals in cancer cells is becoming apparent in some cancers. Better characterisation of the three-dimensional (3D) architecture of solid tumours by utilising 3D cell culture could provide an environment that more closely recapitulates in vivo human tumours for investigating tumour-intrinsic PD-L1 signalling and immunotherapy responses. The role of PD-L1 and how approved immunotherapies may influence its role needs to be fully explored in all cancer types using in vitro cell culture systems that better model tumour heterogeneity compared to standard monolayer cell culture. Within this thesis, human breast prostate and colorectal cancer cell lines were firstly characterised for their expression of immune-inhibitory proteins (PD-L1, PD-1 and PD-L2), immunological proteins (DR4, DR5 and Fas) and tumorigenic proteins (CD44 and HIF1α) at basal level in two-dimensional (2D) monolayer cell culture, before being investigated in two different 3D cell culture models (hanging drop and alginate hydrogel beads) of varying in vitro complexity. In doing this, we were able to demonstrate that cancer cells alter their gene and protein expression levels and develop hypoxia in a 3D environment that more closely mimics human in vivo solid tumours. Cancer cells in 3D reduced their expression of death receptors and antigen presenting machinery which would reduce their susceptibility to immune-mediated cell death and could ultimately hinder their response to immunotherapy. Thereafter, we investigated the biological effects of therapeutically approved anti-PD-L1 monoclonal antibody Atezolizumab, before comparing PD-L1 blockade with PD-L1 knockdown in high PD-L1 expressing breast cancer cells cultured in 2D monolayer and 3D cell culture models. PD-L1 blockade using Atezolizumab demonstrated modest effects on breast cancer cell growth, proliferation, viability, and metabolism in our functional assays, but did reduce the phosphorylation of molecules involved in the PI3K/AKT and MAPK/ERK signalling pathways. PD-L1 knockdown, on the other hand, revealed the importance of PD-L1 expression for the spheroid-forming capabilities of breast cancer cells in our 3D cell culture models. PD-L1 knockdown also potentiated the modest biological effects on breast cancer cell growth, proliferation, viability, and metabolism observed by Atezolizumab treatment. Additionally, cytokine modulation of PD-L1 expression was investigated in combination with PD-L1 blockade and PD-L1 knockdown in our studies. Utilising the 3D alginate model for the culture of breast cancer cells revealed a potential benefit of combining cytokines with PD-L1 targeting for the treatment of breast cancer which warrants further investigation. Altogether this thesis provides new insights into: (1) the expression of immunological and tumorigenic proteins by diverse human cancer cells; (2) how PD-L1 blockade with Atezolizumab may influence PD-L1 intrinsic signalling in breast cancer cells; and (3) how PD-L1 may exhibit a pro-tumour role in breast cancer cells, not only in 2D monolayer but for the first time in two different 3D cell culture models.