EU COST Action CA21130 PRESTO 'P2X receptors as therapeutic targets' inaugural meeting report.

The inaugural meeting of the EU COST Action CA21130 PRESTO was held in February 2023, at the University of Ferrara, Italy. Our meeting report provides an overview of PRESTO, a tribute to Professor Jim Wiley, overviews of the talk, and a speaker synopsis that discusses the resources, models, equipment, and techniques available in different lab groups throughout Europe, increasing the prospect of collaborative research.

The Kinesin Gene KIF26B Modulates the Severity of Post-Traumatic Heterotopic Ossification.

The formation of pathological bone deposits within soft tissues, termed heterotopic ossification (HO), is common after trauma. However, the severity of HO formation varies substantially between individuals, from relatively isolated small bone islands through to extensive soft tissue replacement by bone giving rise to debilitating symptoms. The aim of this study was to identify novel candidate therapeutic molecular targets for severe HO. We conducted a genome-wide scan in men and women with HO of varying severity following hip replacement for osteoarthritis. HO severity was dichotomized as mild or severe, and association analysis was performed with adjustment for age and sex. We next confirmed expression of the gene encoded by the lead signal in human bone and in primary human mesenchymal stem cells. We then examined the effect of gene knockout in a murine model of osseous trans-differentiation, and finally we explored transcription factor phosphorylation in key pathways perturbed by the gene. Ten independent signals were suggestively associated with HO severity, with KIF26B as the lead. We subsequently confirmed KIF26B expression in human bone and upregulation upon BMP2-induced osteogenic differentiation in primary human mesenchymal stem cells, and also in a rat tendo-Achilles model of post-traumatic HO. CRISPR-Cas9 mediated knockout of Kif26b inhibited BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression and mineralized nodule formation in a murine myocyte model of osteogenic trans-differentiation. Finally, KIF26B deficiency inhibited ERK MAP kinase activation during osteogenesis, whilst augmenting p38 and SMAD 1/5/8 phosphorylation. Taken together, these data suggest a role for KIF26B in modulating the severity of post-traumatic HO and provide a potential novel avenue for therapeutic translation.

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.

Distinct Concentration-Dependent Molecular Pathways Regulate Bone Cell Responses to Cobalt and Chromium Exposure from Joint Replacement Prostheses.

Systemic cobalt (Co) and chromium (Cr) concentrations may be elevated in patients with metal joint replacement prostheses. Several studies have highlighted the detrimental effects of this exposure on bone cells in vitro, but the underlying mechanisms remain unclear. In this study, we use whole-genome microarrays to comprehensively assess gene expression in primary human osteoblasts, osteoclast precursors and mature resorbing osteoclasts following exposure to clinically relevant circulating versus local periprosthetic tissue concentrations of Co2+ and Cr3+ ions and CoCr nanoparticles. We also describe the gene expression response in osteoblasts on routinely used prosthesis surfaces in the presence of metal exposure. Our results suggest that systemic levels of metal exposure have no effect on osteoblasts, and primarily inhibit osteoclast differentiation and function via altering the focal adhesion and extracellular matrix interaction pathways. In contrast, periprosthetic levels of metal exposure inhibit both osteoblast and osteoclast activity by altering HIF-1α signaling and endocytic/cytoskeletal genes respectively, as well as increasing inflammatory signaling with mechanistic implications for adverse reactions to metal debris. Furthermore, we identify gene clusters and KEGG pathways for which the expression correlates with increasing Co2+:Cr3+ concentrations, and has the potential to serve as early markers of metal toxicity. Finally, our study provides a molecular basis for the improved clinical outcomes for hydroxyapatite-coated prostheses that elicit a pro-survival osteogenic gene signature compared to grit-blasted and plasma-sprayed titanium-coated surfaces in the presence of metal exposure.

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor.

It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. µCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.

Functional polymorphisms in the P2X7 receptor gene are associated with stress fracture injury.

Military recruits and elite athletes are susceptible to stress fracture injuries. Genetic predisposition has been postulated to have a role in their development. The P2X7 receptor (P2X7R) gene, a key regulator of bone remodelling, is a genetic candidate that may contribute to stress fracture predisposition. The aim of this study is to evaluate the putative contribution of P2X7R to stress fracture injury in two separate cohorts, military personnel and elite athletes. In 210 Israeli Defense Forces (IDF) military conscripts, stress fracture injury was diagnosed (n = 43) based on symptoms and a positive bone scan. In a separate cohort of 518 elite athletes, self-reported medical imaging scan-certified stress fracture injuries were recorded (n = 125). Non-stress fracture controls were identified from these cohorts who had a normal bone scan or no history or symptoms of stress fracture injury. Study participants were genotyped for functional SNPs within the P2X7R gene using proprietary fluorescence-based competitive allele-specific PCR assay. Pearson's chi-squared (χ (2)) tests, corrected for multiple comparisons, were used to assess associations in genotype frequencies. The variant allele of P2X7R SNP rs3751143 (Glu496Ala-loss of function) was associated with stress fracture injury, whilst the variant allele of rs1718119 (Ala348Thr-gain of function) was associated with a reduced occurrence of stress fracture injury in military conscripts (P < 0.05). The association of the variant allele of rs3751143 with stress fractures was replicated in elite athletes (P < 0.05), whereas the variant allele of rs1718119 was also associated with reduced multiple stress fracture cases in elite athletes (P < 0.05). The association between independent P2X7R polymorphisms with stress fracture prevalence supports the role of a genetic predisposition in the development of stress fracture injury.

The P2Y13 receptor regulates phosphate metabolism and FGF-23 secretion with effects on skeletal development.

Purinergic signaling mediates many cellular processes, including embryonic development and regulation of endocrine signaling. The ADP P2Y13 receptor is known to regulate bone and stem cells activities, although relatively little is known about its role in bone development. In this study we demonstrate, using contemporary techniques, that deletion of the P2Y13 receptor results in an age-dependent skeletal phenotype that is governed by changes in phosphate metabolism and hormone levels. Neonatal and postnatal (2 wk) P2Y13 receptor-knockout (KO) mice were indistinguishable from their wild-type (WT) littermate controls. A clear bone phenotype was observed in young (4-wk-old) KO mice compared WT controls, with 14% more trabecular bone, 35% more osteoblasts, 73% fewer osteoclasts, and a 17% thicker growth plate. Mature (>10 wk of age) KO mice showed the opposite bone phenotype, with 14% less trabecular bone, 22% fewer osteoblasts, and 10% thinner growth plate. This age-dependent phenotype correlated with serum fibroblast growth factor-23 (FGF-23) and phosphorus levels that were 65 and 16% higher, respectively, in young KO mice but remained unchanged in mature mice. These findings provide novel insights for the role of the P2Y13 receptor in skeletal development via coordination with hormonal regulators of phosphate homeostasis.

Role of the P2Y13 receptor in the differentiation of bone marrow stromal cells into osteoblasts and adipocytes.

Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y13 -deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y13 R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y13 R(+/+) (but not P2Y13 R(-/-) ) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13 R(-/-) BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13 R(-/-) BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13 R(+/+) MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13 R(-/-) mice. In conclusion, our findings indicate that the P2Y13 R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y13 receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis. STEM Cells 2013;31:2747-2758.

Combining PTH(1-34) and mechanical loading has increased benefit to tibia bone mechanics in ovariectomised mice.

Combined treatment with PTH(1-34) and mechanical loading confers increased structural benefits to bone than monotherapies. However, it remains unclear how this longitudinal adaptation affects the bone mechanics. This study quantified the individual and combined longitudinal effects of PTH(1-34) and mechanical loading on the bone stiffness and strength evaluated in vivo with validated micro-finite element (microFE) models. C57BL/6 mice were ovariectomised at 14-week-old and treated either with injections of PTH(1-34), compressive tibia loading or both interventions concurrently. Right tibiae were in vivo microCT-scanned every 2 weeks from 14 until 24-week-old. MicroCT images were rigidly registered to reference tibia and the cortical organ level (whole bone) and tissue level (midshaft) morphometric properties and bone mineral content were quantified. MicroCT images were converted into voxel-based homogeneous, linear elastic microFE models to estimate the bone stiffness and strength. This approach allowed us for the first time to quantify the longitudinal changes in mechanical properties induced by combined treatments in a model of accelerated bone resorption. Both changes of stiffness and strength were higher with co-treatment than with individual therapies, consistent with increased benefits with the tibia bone mineral content and cortical area, properties strongly associated with the tibia mechanics. The longitudinal data shows that the two bone anabolics, both individually and combined, had persistent benefit on estimated mechanical properties, and that benefits (increased stiffness and strength) remained after treatment was withdrawn.

The RNA binding proteins LARP4A and LARP4B promote sarcoma and carcinoma growth and metastasis.

RNA-binding proteins (RBPs) are emerging as important regulators of cancer pathogenesis. We reveal that the RBPs LARP4A and LARP4B are differentially overexpressed in osteosarcoma and osteosarcoma lung metastases, as well as in prostate cancer. Depletion of LARP4A and LARP4B reduced tumor growth and metastatic spread in xenografts, as well as inhibiting cell proliferation, motility, and migration. Transcriptomic profiling and high-content multiparametric analyses unveiled a central role for LARP4B, but not LARP4A, in regulating cell cycle progression in osteosarcoma and prostate cancer cells, potentially through modulating key cell cycle proteins such as Cyclins B1 and E2, Aurora B, and E2F1. This first systematic comparison between LARP4A and LARP4B assigns new pro-tumorigenic functions to LARP4A and LARP4B in bone and prostate cancer, highlighting their similarities while also indicating distinct functional differences. Uncovering clear biological roles for these paralogous proteins provides new avenues for identifying tissue-specific targets and potential druggable intervention.

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 strategy and clinical relevance of in vitro models of MAP resistance in osteosarcoma: a systematic review.

Over the last 40 years osteosarcoma (OS) survival has stagnated with patients commonly resistant to neoadjuvant MAP chemotherapy involving high dose methotrexate, adriamycin (doxorubicin) and platinum (cisplatin). Due to the rarity of OS, the generation of relevant cell models as tools for drug discovery is paramount to tackling this issue. Four literature databases were systematically searched using pre-determined search terms to identify MAP resistant OS cell lines and patients. Drug exposure strategies used to develop cell models of resistance and the impact of these on the differential expression of resistance associated genes, proteins and non-coding RNAs are reported. A comparison to clinical studies in relation to chemotherapy response, relapse and metastasis was then made. The search retrieved 1891 papers of which 52 were relevant. Commonly, cell lines were derived from Caucasian patients with epithelial or fibroblastic subtypes. The strategy for model development varied with most opting for continuous over pulsed chemotherapy exposure. A diverse resistance level was observed between models (2.2-338 fold) with 63% of models exceeding clinically reported resistance levels which may affect the expression of chemoresistance factors. In vitro p-glycoprotein overexpression is a key resistance mechanism; however, from the available literature to date this does not translate to innate resistance in patients. The selection of models with a lower fold resistance may better reflect the clinical situation. A comparison of standardised strategies in models and variants should be performed to determine their impact on resistance markers. Clinical studies are required to determine the impact of resistance markers identified in vitro in poor responders to MAP treatment, specifically with respect to innate and acquired resistance. A shift from seeking disputed and undruggable mechanisms to clinically relevant resistance mechanisms may identify key resistance markers that can be targeted for patient benefit after a 40-year wait.

YBX1-interacting small RNAs and RUNX2 can be blocked in primary bone cancer using CADD522.

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3' ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3' UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.

A Systematic Review of the Expression, Signalling and Function of P2 Receptors in Primary Bone Cancer.

Primary bone cancers are rare malignant diseases with significant morbidity and mortality. The treatment regimen relies on a combination of surgery (often involving amputation), chemotherapy and radiotherapy with outcomes dependent on localization of the tumour, grade, size and response to chemotherapy. Both treatment options and survival statistics have remained constant over the past 40 years and alternative therapies need to be explored. Purinergic signalling involving the interaction of extracellular nucleotides with P2 receptors has been investigated in numerous cancers with activation or inhibition a topic of debate. To assess whether purinergic signalling could be a viable target in primary bone cancer a systematic review for relevant primary literature published in PubMed, MEDLINE and Web of Science was performed. Search terms were formulated around three separate distinct topics; expression of P2 receptors in primary bone cancer models, P2 receptor signalling pathways involved and the functional consequences of P2 receptor signalling. Searching identified 30 primary articles after screening and eligibility assessments. This review highlights the diverse expression, signalling pathways and functional roles associated with different P2 receptors in primary bone cancers and provides a systematic summary of which P2 receptors are exciting targets to treat primary bone cancer and its associated symptoms.

Absence of P2Y2 Receptor Does Not Prevent Bone Destruction in a Murine Model of Muscle Paralysis-Induced Bone Loss.

Increased incidence of bone fractures in the elderly is associated with gradual sarcopenia. Similar deterioration of bone quality is seen with prolonged bed rest, spinal cord injuries or in astronauts exposed to microgravity and, preceded by loss of muscle mass. Signaling mechanisms involving uridine-5'-triphosphate (UTP) regulate bone homeostasis via P2Y2 receptors on osteoblasts and osteoclasts, whilst dictating the bone cells' response to mechanical loading. We hypothesized that muscle paralysis-induced loss of bone quality would be prevented in P2Y2 receptor knockout (KO) mice. Female mice injected with botulinum toxin (BTX) in the hind limb developed muscle paralysis and femoral DXA analysis showed reduction in bone mineral density (<10%), bone mineral content (<16%) and bone area (<6%) in wildtype (WT) compared to KO littermates (with <13%, <21%, <9% respectively). The femoral metaphyseal strength was reduced equally in both WT and KO (<37%) and <11% in diaphysis region of KO, compared to the saline injected controls. Tibial micro-CT showed reduced cortical thickness (12% in WT vs. 9% in KO), trabecular bone volume (38% in both WT and KO), trabecular thickness (22% in WT vs. 27% in KO) and increased SMI (26% in WT vs. 19% in KO) after BTX. Tibial histomorphometry showed reduced formation in KO (16%) but unchanged resorption in both WT and KO. Furthermore, analyses of DXA and bone strength after regaining the muscle function showed partial bone recovery in the KO but no difference in the bone recovery in WT mice. Primary osteoblasts from KO mice displayed increased viability and alkaline phosphatase activity but, impaired bone nodule formation. Significantly more TRAP-positive osteoclasts were generated from KO mice but displayed reduced resorptive function. Our data showed that hind limb paralysis with a single dose of BTX caused profound bone loss after 3 weeks, and an incomplete reversal of bone loss by week 19. Our findings indicate no role of the P2Y2 receptor in the bone loss after a period of skeletal unloading in mice or, in the bone recovery after restoration of muscle function.

A novel prostaglandin E receptor 4 (EP4) small molecule antagonist induces articular cartilage regeneration.

Articular cartilage repair and regeneration is an unmet clinical need because of the poor self-regeneration capacity of the tissue. In this study, we found that the expression of prostaglandin E receptor 4 (PTGER4 or EP4) was largely increased in the injured articular cartilage in both humans and mice. In microfracture (MF) surgery-induced cartilage defect (CD) and destabilization of the medial meniscus (DMM) surgery-induced CD mouse models, cartilage-specific deletion of EP4 remarkably promoted tissue regeneration by enhancing chondrogenesis and cartilage anabolism, and suppressing cartilage catabolism and hypertrophy. Importantly, knocking out EP4 in cartilage enhanced stable mature articular cartilage formation instead of fibrocartilage, and reduced joint pain. In addition, we identified a novel selective EP4 antagonist HL-43 for promoting chondrocyte differentiation and anabolism with low toxicity and desirable bioavailability. HL-43 enhanced cartilage anabolism, suppressed catabolism, prevented fibrocartilage formation, and reduced joint pain in multiple pre-clinical animal models including the MF surgery-induced CD rat model, the DMM surgery-induced CD mouse model, and an aging-induced CD mouse model. Furthermore, HL-43 promoted chondrocyte differentiation and extracellular matrix (ECM) generation, and inhibited matrix degradation in human articular cartilage explants. At the molecular level, we found that HL-43/EP4 regulated cartilage anabolism through the cAMP/PKA/CREB/Sox9 signaling. Together, our findings demonstrate that EP4 can act as a promising therapeutic target for cartilage regeneration and the novel EP4 antagonist HL-43 has the clinical potential to be used for cartilage repair and regeneration.

RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis.

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and ß-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor-related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties.

Background: Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for the development of novel therapeutic strategies. Here, we report in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (P2RX7B), in OS progression and metastasis. Methods: TE85 and MNNG-HOS OS cells were transfected with P2RX7B. These cell lines were then characterised and assessed for proliferation, cell adhesion, migration and invasion in vitro. We used these cells to perform both paratibial and tail vein injected mouse studies where the primary tumour, bone and lungs were analysed. We used RNA-seq to identify responsive pathways relating to P2RX7B. Results: Our data shows that P2RX7B expression confers a survival advantage in TE85 + P2RX7B and MNNG-HOS + P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, demonstrating a metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS + P2RX7B tumours exhibited cancer-associated ectopic bone formation that was abrogated with A740003 treatment. A pro-metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of tumour cells to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. Conclusion: Our data illustrates a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a future therapeutic target in human OS.

Septoclast deficiency accompanies postnatal growth plate chondrodysplasia in the toothless (tl) osteopetrotic, colony-stimulating factor-1 (CSF-1)-deficient rat and is partially responsive to CSF-1 injections.

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.