The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment

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

Tissue engineering laboratory models of the small intestine.

In recent years, three-dimensional (3D) cell culture models of the small intestine have gained much attention. These models support cell proliferation, migration, and differentiation, and encourage tissue organization which is not possible in two-dimensional (2D) culture systems. Furthermore, the use of a wide variety of cell culture scaffolds and support substrates have revealed considerable differences in cell behavior and tissue organization. These systems have been used in combination with intestinal stem cells, organoid units or human colonic adenocarcinoma cell lines such as Caco-2 and HT29-MTX to generate a number of in vitro and in vivo models of the intestine. Here, we review the current 2D and 3D tissue engineering models of the intestine to determine the most effective sources of intestinal cells and current research on support scaffolds capable of inducing the morphological architecture and function of the intestinal mucosa

Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium

The human intestinal cell lines: Caco-2 and HT29-MTX cells have been used extensively in 2D and 3D cell cultures as simple models of the small intestinal epithelium in vitro. This study aimed to investigate the potential of three hydrogel scaffolds to support the 3D culture of Caco-2 and HT29-MTX cells and critically assess their use as scaffolds to stimulate villi formation to model a small intestinal epithelium in vitro. Here, alginate, l-pNIPAM, and l-pNIPAM-co-DMAc hydrogels were investigated. The cells were suspended within or layered on these hydrogels and maintained under static or dynamic culture conditions for up to 21days. Caco-2 cell viability was increased when layered on the synthetic hydrogel scaffolds, but reduced when suspended within the synthetic hydrogels. In contrast, HT29-MTX cells remained viable when suspended within or layered on all 3D cultures. Interestingly, cells cultured in and on the alginate hydrogel scaffolds formed multilayer spheroid structures, whilst the cells layered on synthetic hydrogels formed villus-like structures. Immunohistochemistry staining demonstrated positive expression of enterocyte differentiation markers and goblet cell marker. In conclusion, l-pNIPAM hydrogel scaffolds supported both cell lines and induced formation of villus-like structures when cells were layered on and cultured under dynamic conditions. The ability of the l-pNIPAM to recapitulate the 3D structure and differentiate main cell types of human intestinal villi may deliver a potential alternative in vitro model for studying intestinal disease and for drug testing. Forty percent of hospital referrals are linked to disorders of the digestive tract. Current studies have utilised animal models or simple cultures of isolated cells which do not behave in the same manner as human intestine. Thus new models are required which more closely mimic the behaviour of intestinal cells. Here, we tested a number of scaffolds and conditions to develop a cell culture model which closely represents the 3D environment seen within the human small intestine. We successfully created structures seen within the intestine which have not previously been possible with other culture models. These models could be used to investigate tissue engineering, drug discovery, and used asan alternative to in vivo animal models in drug toxicity studies. [Abstract copyright: Copyright © 2017. Published by Elsevier Ltd.

Assessment of ferroptosis inducers and Nrf2 inhibitors as radiosensitisers in 2D and 3D breast cancer cell cultures

Ferroptosis is a form of programmed cell death that is modulated in some cancer cells as a pro-survival mechanism. Induction of ferroptosis is a potential anti-cancer strategy, and enhancement of ferroptosis using ferroptosis inducers has the potential to enhance current anti-tumour mechanisms. In this study, we assessed the effect of the ferroptosis inducers Erastin, RSL-3 and FIN-56 on radiosensitivity in 2D cell culture, and in 3D alginate tumour spheroids from breast cancer cell lines. Since some tumours modulate ferroptosis via increased Nrf2 production, and MCF-7 and MDA-MB-231 both produce Nrf2 protein, we also assessed the effects of the Nrf2 inhibitor ML385 on radiosensitivity. MDA-MB-231 was highly sensitive to all ferroptosis inducers, and ferroptosis was reversed by the ferroptosis inhibitors Ferrostatin-1, Liproxstatin-1 and Deferoxamine. MCF-7 was resistant to all ferroptosis inducers. MDA-MB-231 and MCF-7 cells were sensitive to irradiation in 2D cell culture but resistant to irradiation in 3D alginate spheroids. Ferroptosis inducers did not synergistically enhance irradiation-induced cell death in 2D cell cultures. There was also no robust enhancement to irradiation effects with ferroptosis inducers in 2D or 3D cell culture. Ferroptosis inducers did, however, show a heterogeneous response in 3D cell culture, in that isogenic spheroids responded differently within the same spheroid. The Nrf2 inhibitor ML385 showed no synergistic enhancement of ferroptotic cell death when combined with irradiation. These studies suggest targeting ferroptosis does not induce short-term enhancement of ferroptotic cell death

Molecular Action of Polyphenols in Leukaemia and Their Therapeutic Potential

Leukaemia is a malignant disease of the blood. Current treatments for leukaemia are associated with serious side-effects. Plant-derived polyphenols have been identified as potent anti-cancer agents and have been shown to work synergistically with standard chemotherapy agents in leukaemia cell lines. Polyphenols have multiple mechanisms of action and have been reported to decrease cell proliferation, arrest cell cycle and induce apoptosis via the activation of caspase (3, 8 and 9); the loss of mitochondrial membrane potential and the release of cytochrome c. Polyphenols have been shown to suppress activation of transcription factors, including NF-kB and STAT3. Furthermore, polyphenols have pro-oxidant properties, with increasing evidence that polyphenols inhibit the antioxidant activity of glutathione, causing oxidative DNA damage. Polyphenols also induce autophagy-driven cancer cell death and regulate multidrug resistance proteins, and thus may be able to reverse resistance to chemotherapy agents. This review examines the molecular mechanism of action of polyphenols and discusses their potential therapeutic targets. Here, we discuss the pharmacological properties of polyphenols, including their anti-inflammatory, antioxidant, anti-proliferative, and anti-tumour activities, and suggest that polyphenols are potent natural agents that can be useful therapeutically; and discuss why data on bioavailability, toxicity and metabolism are essential to evaluate their clinical use

Programmed death-ligand 1 expression in human cancer cell lines in two-dimensional and three-dimensional cell culture systems

Solid tumours are characterised by a three-dimensional (3D) architecture that provides specific survival advantages such as resistance to anti-cancer drugs. The expression of programmed death-ligand 1 (PD-L1) is one such survival mechanism employed by tumours to mediate immune evasion, drug resistance and tumour progression. Here we investigated whether the expression of PD-L1 by human cancer cell lines altered in a 3D cell culture setting as opposed to their two-dimensional (2D) monolayer counterparts. We utilised well-established 3D cell culturing systems that facilitate the formation of spheroids and display heterogeneous populations of cells resembling that found in the tumour microenvironment, to assess PD-L1 expression. We found that PD-L1 expression changed in human breast, prostate and colorectal cancer cell lines in 3D cell culture systems when compared to their 2D counterparts. The level of expression of PD-L1 by tumour cells in 3D cell culture is more likely to mimic that of an in vivo tumour microenvironment than 2D cell culture, and may better able the investigation of the tumour-intrinsic role of PD-L1

Use of l-pNIPAM hydrogel as a 3D-scaffold for intestinal crypts and stem cell tissue engineering

Intestinal stem cells hold great potential in tissue regeneration of the intestine, however, there are key limitations in their culture in vitro. We previously reported a novel synthetic non-biodegradable hydrogel as a 3D culture model for intestinal epithelium using Caco2 and HT29-MTX cells. Here, we investigated the potential of this system as a 3D scaffold for crypts and single intestinal stem cells to support long-term culture and differentiation. Intestinal crypts were extracted from murine small intestines and Lgr5+ stem cells isolated by magnetic activated cell sorting. Crypts and stem cells were suspended within Matrigel or L-pNIPAM for 14 days or suspended within Matrigel for 7 days then released, dissociated, and suspended within, or on L-pNIPAM hydrogel for 28 days. Cellular behaviour and phenotype were determined by histology and immunohistochemistry for stem cell and differentiation markers: Lgr5, E-cadherin MUC2 chromograninA and lysozymes. Isolated crypts and Lgr5+ intestinal stem cells formed enteroids with a central lumen surrounded by multiple crypt-like buds when cultured in Matrigel. In contrast, when crypts and stem cells were directly suspended within, or layered on L-pNIPAM hydrogel under dynamic culture conditions they formed spherical balls of cells, with no central lumen. When enteroids were initially formed in Matrigel from crypts or single Lgr5+ intestinal stem cells and dissociated into small fragments or single cells and transferred to L-pNIPAM hydrogel they formed new larger enteroids with numerous crypt-like buds. These crypt-like buds showed the presence of mucin-producing cells, which resembled goblet cells, scattered throughout their structures. Immunohistochemistry staining also showed the expression of Lgr5 and differentiation markers of all the main intestinal cell types including: enterocytes, goblet cells, enteroendocrine and Paneth cells. This demonstrated that L-pNIPAM hydrogel supported long-term culture of crypts and Lgr5+ stem cells and promoted intestinal cell differentiation

Polyphenols are responsible for the proapoptotic properties of pomegranate juice on leukemia cell lines

Pomegranates have shown great promise as anti-cancer agents in a number of cancers including clinical trials in prostate cancer. We have previously shown pomegranate juice (PGJ) induced apoptosis and preferentially alters the cell cycle in leukemia cell lines compared with nontumor control cells. However, the agents responsible have not yet been fully elucidated. Treatment of four leukemia cell lines with five fractions obtained from PGJ by solid phase extraction demonstrated that only the acetonitrile fractions decreased adenosine triphosphate (ATP) levels in all leukemia cell lines. Acetonitrile fractions also significantly activated caspase-3 and induced nuclear morphology characteristic of apoptosis. S phase arrest was induced by acetonitrile fractions which matched S phase arrest seen previously following whole PGJ treatments. The acetonitrile fractions contained higher phenol content than whole PGJ whereas only low levels of phenols were seen in any other fraction. Liquid chromatography mass spectrometry (LC–MS) analysis demonstrated that acetonitrile fractions were enriched in ellagitannins, ellagic acid, and hydroxycinnamic acid derivatives but depleted in anthocyanins. Individual treatments with identified compounds demonstrated that the ellagitannin: punicalagin was the most active and mimicked the responses seen following acetonitrile fraction treatment. Bioactive components within pomegranate were confined to the acetonitrile fraction of PGJ. The enrichment in ellagitannins and hydroxycinnamic acids suggest these may provide the majority of the bioactivities of PGJ. Individual treatments with compounds identified demonstrated that the ellagitannin: punicalagin was the most active agent, highlighting this compound as a key bioactive agent in PGJ

Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration

OBJECTIVES: Interleukin 1 (IL-1) is potentially important in the pathogenesis of intervertebral disc (IVD) degeneration; increasing production of matrix degradation enzymes and inhibiting matrix synthesis. Although IL-1 polymorphisms have been linked to increased risk of IVD degeneration, it is still unclear whether IL-1 drives IVD degeneration in vivo or is a secondary feature of degeneration. Here, we investigated whether IVD degeneration could be induced spontaneously by the removal of the natural inhibitor of IL-1 (IL-1 receptor antagonist) in mice that lack a functional IL-1rn gene. METHODS: Histological staining and immunohistochemistry was performed on BALB/c IL-1rn(+/+) and IL-1rn(-/-) mice to examine degeneration and to localise and detect IL-1, matrix metalloproteinases (MMP)3, MMP7, a disintigrin and MMP with thrombospondin motifs (ADAMTS)4 protein production. In addition, IVD cells were isolated using collagenase and proliferation potential determined. RESULTS: IL-1rn(-/-) knockout mice displayed typical features of human disc degeneration: loss of proteoglycan and normal collagen structure and increased expression of matrix degrading enzymes: MMP3; MMP7 and ADAMTS4. Histological grade of degeneration increased in IL-1rn(-/-) mice which was more evident within older mice. In addition IVD cells isolated from IL-1rn(-/-) mice displayed reduced proliferation potential. CONCLUSIONS: Here, we show that IL-1rn(-/-) mice develop spinal abnormalities that resemble characteristic features associated with human disc degeneration. The current evidence is consistent with a role for IL-1 in the pathogenesis of IVD degeneration. The imbalance between IL-1 and IL-1Ra which is observed during human IVD degeneration could therefore be a causative factor in the degeneration of the IVD, and as such, is an appropriate pharmaceutical target for inhibiting degeneration.</p