Ablation of the dystrophin Dp71f alternative C-terminal variant increases sarcoma tumour cell aggressiveness.

Alterations in Dp71 expression, the most ubiquitous dystrophin isoform, have been associated with patient survival across tumours. Intriguingly, in certain malignancies, Dp71 acts as a tumour suppressor, while manifesting oncogenic properties in others. This diversity could be explained by the expression of two Dp71 splice variants encoding proteins with distinct C-termini, each with specific properties. Expression of these variants has impeded the exploration of their unique roles. Using CRISPR/Cas9, we ablated the Dp71f variant with the alternative C-terminus in a sarcoma cell line not expressing the canonical C-terminal variant, and conducted molecular (RNAseq) and functional characterisation of the knockout cells. Dp71f ablation induced major transcriptomic alterations, particularly affecting the expression of genes involved in calcium signalling and ECM-receptor interaction pathways. The genome-scale metabolic analysis identified significant downregulation of glucose transport via membrane vesicle reaction (GLCter) and downregulated glycolysis/gluconeogenesis pathway. Functionally, these molecular changes corresponded with, increased calcium responses, cell adhesion, proliferation, survival under serum starvation and chemotherapeutic resistance. Knockout cells showed reduced GLUT1 protein expression, survival without attachment and their migration and invasion in vitro and in vivo were unaltered, despite increased matrix metalloproteinases release. Our findings emphasise the importance of alternative splicing of dystrophin transcripts and underscore the role of the Dp71f variant, which appears to govern distinct cellular processes frequently dysregulated in tumour cells. The loss of this regulatory mechanism promotes sarcoma cell survival and treatment resistance. Thus, Dp71f is a target for future investigations exploring the intricate functions of specific DMD transcripts in physiology and across malignancies.

Investigating the Involvement of C-X-C Motif Chemokine 5 and P2X7 Purinoceptor in Ectopic Calcification in Mouse Models of Duchenne Muscular Dystrophy.

Ectopic calcification of myofibers is an early pathogenic feature in patients and animal models of Duchenne muscular dystrophy (DMD). In previous studies using the Dmdmdx-ßgeo mouse model, we found that the dystrophin-null phenotype exacerbates this abnormality and that mineralised myofibers are surrounded by macrophages. Furthermore, the P2X7 purinoceptor, functioning in immune cells offers protection against dystrophic calcification. In the present study, by exploring transcriptomic data from Dmdmdx mice, we hypothesised these effects to be mediated by C-X-C motif chemokine 5 (CXCL5) downstream of P2X7 activation. We found that CXCL5 is upregulated in the quadriceps muscles of Dmdmdx-ßgeo mice compared to wild-type controls. In contrast, at the cell level, dystrophic (SC5) skeletal muscle cells secreted less CXCL5 chemokine than wild-type (IMO) controls. Although release from IMO cells was increased by P2X7 activation, this could not explain the elevated CXCL5 levels observed in dystrophic muscle tissue. Instead, we found that CXCL5 is released by dystrophin-null macrophages in response to P2X7 activation, suggesting that macrophages are the source of CXCL5 in dystrophic muscles. The effects of CXCL5 upon mineralisation were investigated using the Alizarin Red assay to quantify calcium deposition in vitro. In basal (low phosphate) media, CXCL5 increased calcification in IMO but not SC5 myoblasts. However, in cultures treated in high phosphate media, to mimic dysregulated phosphate metabolism occurring in DMD, CXCL5 decreased calcification in both IMO and SC5 cells. These data indicate that CXCL5 is part of a homoeostatic mechanism regulating intracellular calcium, that CXCL5 can be released by macrophages in response to the extracellular ATP damage-associated signal, and that CXCL5 can be part of a damage response to protect against ectopic calcification. This mechanism is affected by DMD gene mutations.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

Established Models and New Paradigms for Hypoxia-Driven Cancer-Associated Bone Disease.

The five-year survival rate for primary bone cancers is ~ 70% while almost all cases of secondary metastatic bone cancer are terminal. Hypoxia, the deficiency of oxygen which occurs as the rate of tumour growth exceeds the supply of vascularisation, is a key promoter of tumour progression. Hypoxia-driven effects in the primary tumour are wide ranging including changes in gene expression, dysregulation of signalling pathways, resistance to chemotherapy, neovascularisation, increased tumour cell proliferation and migration. Paget's seed and soil theory states that for a metastasising tumour cell 'the seed' it requires the correct microenvironment 'soil' to colonise. Why and how metastasising tumour cells colonise the bone is a complex and intriguing problem. However, once present tumour cells are able to disrupt bone homeostasis through increasing osteoclast activity and downregulating osteoblast function. Osteoclast resorption releases growth factors from the bone matrix that subsequently contribute to the proliferation of invasive tumour cells creating the vicious cycle of bone loss and metastatic cancer progression. Recently, we have shown that hypoxia increases expression and release of lysyl oxidase (LOX) from primary mammary tumours, which in turn disrupts bone homeostasis to favour osteolytic degradation to create pre-metastatic niches in the bone microenvironment. We also demonstrated how treatment with bisphosphonates could block this cancer-induced bone remodelling and reduce secondary bone metastases. This review describes the roles of hypoxia in primary tumour progression to metastasis, with a focus on key signalling pathways and treatment options to reduce patient morbidity and increase survival.

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

Generation of a novel mouse model for the inducible depletion of macrophages in vivo.

Macrophages play an essential role in tissue homeostasis, innate immunity, inflammation, and wound repair. Macrophages are also essential during development, severely limiting the use of mouse models in which these cells have been constitutively deleted. Consequently, we have developed a transgenic model of inducible macrophage depletion in which macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline. Induction of the DTA protein in transgenic animals resulted in a significant 50% reduction in CD68+ macrophages of the liver, spleen, and bone over a period of 6 weeks. Pertinently, the macrophages remaining after doxycycline treatment were substantially smaller and are functionally impaired as shown by reduced inflammatory cytokine production in response to lipopolysaccharide. This inducible model of macrophage depletion can now be utilized to determine the role of macrophages in both development and animal models of chronic inflammatory diseases.

The P2X7 purinoceptor in pathogenesis and treatment of dystrophino- and sarcoglycanopathies.

Dystrophinopathy and sarcoglycanopathies are incurable diseases caused by mutations in the genes encoding dystrophin or members of the dystrophin associated protein complex (DAPC). Restoration of the missing dystrophin or sarcoglycans via genetic approaches is complicated by the downsides of personalised medicines and immune responses against re-expressed proteins. Thus, the targeting of disease mechanisms downstream from the mutant protein has a strong translational potential. Acute muscle damage causes release of large quantities of ATP, which activates P2X7 purinoceptors, resulting in inflammation that clears dead tissues and triggers regeneration. However, in dystrophic muscles, loss of α-sarcoglycan ecto-ATPase activity further elevates extracellular ATP (eATP) levels, exacerbating the pathology. Moreover, seemingly compensatory P2X7 upregulation in dystrophic muscle cells, combined with high eATP leads to further damage. Accordingly, P2X7 blockade alleviated dystrophic damage in mouse models of both dystrophinopathy and sarcoglycanopathy. Existing P2X7 blockers could be re-purposed for the treatment of these highly debilitating diseases.

P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles.

Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells. To investigate the role of P2X7 in dystrophic calcification, we utilised the Dmd mdx-ßgeo dystrophin-null mouse model of DMD crossed with a global P2X7 knockout (P2rx7 -/- ) or with our novel P2X7 knockin-knockout mouse (P2x7 KiKo ), which expresses P2X7 in macrophages but not muscle cells. Total loss of P2X7 increased EC, indicating that P2X7 overexpression is a protective mechanism against dystrophic mineralisation. Given that muscle-specific P2X7 ablation did not affect dystrophic EC, this underlined the role of P2X7 receptor expression on the inflammatory cells. Serum phosphate reflected dystrophic calcification, with the highest serum phosphate levels found in genotypes with the most ectopic mineral. To further investigate the underlying mechanisms, we measured phosphate release from cells in vitro, and found that dystrophic myoblasts released less phosphate than non-dystrophic cells. Treatment with P2X7 antagonists increased phosphate release from both dystrophic and control myoblasts indicating that muscle cells are a potential source of secreted phosphate while macrophages protect against ectopic mineralisation. Treatment of cells with high phosphate media engendered mineral deposition, which was decreased in the presence of the P2X7 agonist BzATP, particularly in cultures of dystrophic cells, further supporting a protective role for P2X7 against ectopic mineralisation in dystrophic muscle.

Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties.

Knockout and Knock-in Mouse Models to Study Purinergic Signaling.

Purinergic signaling involves extracellular purines and pyrimidines acting upon specific cell surface purinoceptors classified into the P1, P2X, and P2Y families for nucleosides and nucleotides. This widespread signaling mechanism is active in all major tissues and influences a range of functions in health and disease. Orthologs to all but one of the human purinoceptors have been found in mouse, making this laboratory animal a useful model to study their function. Indeed, analyses of purinoceptors via knock-in or knockout approaches to produce gain or loss of function phenotypes have revealed several important therapeutic targets. None of the homozygous purinoceptor knockouts proved to be developmentally lethal, which suggest that either these receptors are not involved in key developmental processes or that the large number of receptors in each family allowed for functional compensation. Different models for the same purinoceptor often show compatible phenotypes but there have been examples of significant discrepancies. These revealed unexpected differences in the structure of human and mouse genes and emphasized the importance of the genetic background of different mouse strains. In this chapter, we provide an overview of the current knowledge and new trends in the modifications of purinoceptor genes in vivo. We discuss the resulting phenotypes, their applications and relative merits and limitations of mouse models available to study purinoceptor subtypes.

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair.

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like' printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development.

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading.

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y(13) receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y(13) receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild-type (WT) and P2Y(13) receptor knockout (P2Y(13)R-/-) mice were subject to non-invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro-computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y(13)R-/- mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y(13)R-/- mice compared with controls. In vitro, primary P2Y(13)R-/- osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y(13)R-/- osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto-enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y(13) receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX-induced bone loss after P2Y(13) receptor depletion as previously described, suggests a potential role for P2Y(13) receptor antagonist-based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.

Purinergic signalling in bone.

Purinergic signaling in bone was first proposed in the early 1990s with the observation that extracellular ATP could modulate events crucial to the normal functioning of bone cells. Since then the expression of nearly all the P2Y and P2X receptors by osteoblasts and osteoclasts has been reported, mediating multiple processes including cell proliferation, differentiation, function, and death. This review will highlight the most recent developments in the field of purinergic signaling in bone, with a special emphasis on recent work resulting from the European Framework 7 funded collaboration ATPBone, as well as Arthritis Research UK and Bone Research Society supported projects.

Isolation and culture of human osteoblasts.

The skeleton is a dynamic organ that is constantly active throughout life. The highly coordinated actions of bone cells early in life determine the body's shape and form, whilst the constant remodelling (bone resorption followed by an equal amount of bone formation) during adulthood helps to maintain skeletal mass and repair microdamage. When the balance of bone resorption and bone formation becomes unequal, bone diseases, such as osteoporosis, occur. In order to develop drugs to combat bone disease, it is important to know the regulatory systems involved in normal bone formation and resorption. In this chapter, we concentrate on bone formation, providing a detailed guide to isolating and culturing primary human osteoblasts in bone explant cultures, as well as the methodology used to characterise and monitor the function of osteoblasts. In combination, these methods provide a powerful tool in bone cell biology and in the development of new novel treatments for bone disease.

Purinergic signalling in osteoblasts.

The skeleton is maintained throughout life via the finely tuned actions of osteoblasts and osteoclasts, with disruption in this balance eventually leading to bone disease. The exact mechanisms balancing these actions are not fully known, although several regulatory systems are known to be involved. The involvement of purinergic signalling in bone has come to light over the past 20 years or so. This review will highlight the current knowledge of purinergic signalling in osteoblasts - covering expression of P2 receptors, mechanisms of ATP release and degradation, P2 receptor mediated signalling and finally the functional consequences of P2 receptor signalling in bone.