Programmed death-ligand 1 (PD-L1) is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity. Many tumors exploit this mechanism by overexpressing PD-L1 which often correlates with poor prognosis. Some tumors have also recently been shown to express PD-1. On tumors, PD-L1 binding to PD-1 on immune cells promotes immune evasion and tumor progression, primarily by inhibition of cytotoxic T lymphocyte effector function. PD-1/PD-L1-targeted therapy has revolutionized the cancer therapy landscape and has become the first-line treatment for some cancers, due to their ability to promote durable anti-tumor immune responses in select patients with advanced cancers. Despite this clinical success, some patients have shown to be unresponsive, hyperprogressive or develop resistance to PD-1/PD-L1-targeted therapy. The exact mechanisms for this are still unclear. This review will discuss the current status of PD-1/PD-L1-targeted therapy, oncogenic expression of PD-L1, the new and emerging tumor-intrinisic roles of PD-L1 and its receptor PD-1 and how they may contribute to tumor progression and immunotherapy responses as shown in different oncology models.

Multiple myeloma is a plasma cell malignancy that causes debilitating bone disease and fractures, in which TGFβ plays a central role. Current treatments do not repair existing damage and fractures remain a common occurrence. We developed a novel low tumor phase murine model mimicking the plateau phase in patients as we hypothesized this would be an ideal time to treat with a bone anabolic. Using in vivo μCT we show substantial and rapid bone lesion repair (and prevention) driven by SD‐208 (TGFβ receptor I kinase inhibitor) and chemotherapy (bortezomib and lenalidomide) in mice with human U266‐GFP‐luc myeloma. We discovered that lesion repair occurred via an intramembranous fracture repair‐like mechanism and that SD‐208 enhanced collagen matrix maturation to significantly improve fracture resistance. Lesion healing was associated with VEGFA expression in woven bone, reduced osteocyte‐derived PTHrP, increased osteoblasts, decreased osteoclasts, and lower serum tartrate‐resistant acid phosphatase 5b (TRACP‐5b). SD‐208 also completely prevented bone lesion development in mice with aggressive JJN3 tumors, and was more effective than an anti‐TGFβ neutralizing antibody (1D11). We also discovered that SD‐208 promoted osteoblastic differentiation (and overcame the TGFβ‐induced block in osteoblastogenesis) in myeloma patient bone marrow stromal cells in vitro, comparable to normal donors. The improved bone quality and fracture‐resistance with SD‐208 provides incentive for clinical translation to improve myeloma patient quality of life by reducing fracture risk and fatality. © 2019 American Society for Bone and Mineral Research.

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, hyper-progressive or develop resistance. Better characterisation of the 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. Here we investigated whether PD-L1 expression by human breast, prostate and colorectal cancer cell lines altered in 3D cell culture models compared to their 2D monolayer counterparts. We found that PD-L1 expression changed in 3D-cultured cancer cells when compared to 2D-cultured cells. Additionally, the expression of immunological markers, PD-1, PD-L2, CD44, DR4, DR5, Fas, and HLA-ABC were assessed in 3D cell culture and compared to their expression in 2D. These markers were also altered in 3D compared to 2D-cultured cells, highlighting the importance of utilising 3D models which may better able the investigation of tumour-intrinsic PD-L1 signalling, responses to PD-1/PD-L1-targeted therapy, and combination therapies.

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Tumorigenic expression of programmed death-ligand 1 (PD-L1) and its receptor PD-1 is found in many cancers, though the intrinsic roles of these proteins and how immunotherapy treatment may affect these pathways needs to be fully explored across human cancers. One platform to assess the expression and regulation of these proteins is 3D cell culture, which is thought to more closely mimic the architecture of solid tumors and more closely recapitulate in vivo human tumors in terms of cell heterogeneity, immunological and tumorigenic marker expression and treatment response, to investigate PD-L1/PD-1 signaling in cancer cells. Human breast (MDA-MB-231 and MCF-7), prostate (LNCaP and PC3) and colorectal (SW480 and SW620) cancer cell lines were cultured in both scaffold-free and scaffold-based 3D cell culture models to facilitate the formation of spheroids that display cell heterogeneity. 3D cancer models were analyzed by microscopy, qPCR and flow cytometry to assess the viability of spheroids, and measure any alterations in immunological and tumorigenic markers at mRNA and protein level, respectively, compared to their 2D monolayer counterparts. Breast, prostate and colorectal cancer cells cultured in 3D were highly viable over time and displayed significantly altered levels of immunological and tumorigenic marker expression at mRNA and/or protein level compared to their 2D monolayer counterparts. Importantly, PD-L1 expression changed in a 3D environment which may be linked to the increased cell-cell and cell-ECM interactions; increased expression of PD-L1 regulatory genes/proteins; or hypoxic regions that establish in 3D spheroids. The significantly altered expression levels of immunological and tumorigenic markers in a 3D cell culture environment is more likely to mimic that of an in vivo human tumor than standard 2D cell culture; allowing for optimized in vitro evaluation of the role of tumorigenic PD-L1/PD-1 and how immunotherapy treatment may affect these pathways. Citation Format: Katie Hudson, Neil Cross, Nicola Jordan-Mahy, Rebecca Leyland. Characterization of 3D models of human breast, prostate and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2988.

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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.