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Ectopic calcification of myofibers is an early pathogenic feature in patients and animal models of Duchenne muscular dystrophy (DMD). In previous studies using the Dmd mdx-βgeo 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 Dmd mdx 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 Dmd mdx-βgeo 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., (© 2024 The Author(s). Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

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., (© The Author(s) 2024. Published by Oxford University Press.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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- βgeo 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rumney, Róg, Chira, Kao, Al-Khalidi and Górecki.)

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., (© 2022. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

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 VEGF 165 protein (rhVEGF 165 ) 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 rhVEGF 165 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-63 VEGF165 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.

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.

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.

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.