Extracellular Adenosine Research Articles

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

Tumor intrinsic and extrinsic functions of CD73 and the adenosine pathway in lung cancer.

The adenosine pathway is an exciting new target in the field of cancer immunotherapy. CD73 is the main producer of extracellular adenosine. Non-small cell lung cancer (NSCLC) has one of the highest CD73 expression signatures among all cancer types and the presence of common oncogenic drivers of NSCLC, such as mutant epidermal growth factor receptor (EGFR) and KRAS, correlate with increased CD73 expression. Current immune checkpoint blockade (ICB) therapies only benefit a subset of patients, and it has proved challenging to understand which patients might respond even with the current understanding of predictive biomarkers. The adenosine pathway is well known to disrupt cytotoxic function of T cells, which is currently the main target of most clinical agents. Data thus far suggests that combining ICB therapies already in the clinic with adenosine pathway inhibitors provides promise for the treatment of lung cancer. However, antigen loss or lack of good antigens limits efficacy of ICB; simultaneous activation of other cytotoxic immune cells such as natural killer (NK) cells can be explored in these tumors. Clinical trials harnessing both T and NK cell activating treatments are still in their early stages with results expected in the coming years. In this review we provide an overview of new literature on the adenosine pathway and specifically CD73. CD73 is thought of mainly for its role as an immune modulator, however recent studies have demonstrated the tumor cell intrinsic properties of CD73 are potentially as important as its role in immune suppression. We also highlight the current understanding of this pathway in lung cancer, outline ongoing studies examining therapies in combination with adenosine pathway targeting, and discuss future prospects.

Characterization of purinergic signaling in tumor-infiltrating lymphocytes from lower- and high-grade gliomas.

Malignant gliomas are highly heterogeneous glia-derived tumors that present an aggressive and invasive nature, with a dismal prognosis. The multi-dimensional interactions between glioma cells and other tumor microenvironment (TME) non-tumoral components constitute a challenge to finding successful treatment strategies. Several molecules, such as extracellular purines, participate in signaling events and support the immunosuppressive TME of glioma patients. The purinergic signaling and the ectoenzymes network involved in the metabolism of these extracellular nucleotides are still unexplored in the glioma TME, especially in lower-grade gliomas (LGG). Also, differences between IDH-mutant (IDH-Mut) versus wild-type (IDH-WT) gliomas are still unknown in this context. For the first time, to our knowledge, this study characterizes the TME of LGG, high-grade gliomas (HGG) IDH-Mut, and HGG IDH-WT patients regarding purinergic ectoenzymes and P1 receptors, focusing on tumor-infiltrating lymphocytes. Here, we show that ectoenzymes from both canonical and non-canonical pathways are increased in the TME when compared to the peripheral blood. We hypothesize this enhancement supports extracellular adenosine generation, hence increasing TME immunosuppression.

DT-0111: a novel P2X3 receptor antagonist.

Extracellular adenosine 5'-triphosphate (ATP) acts as an autocrine and paracrine agent, the actions of which on affected cells are mediated by P2 receptors (P2R), which include trans cell-membrane cationic channels (P2XRs), and G protein coupled receptors (P2YRs). The mammalian P2X receptors form homotrimeric or heterotrimeric cationic channels, each of which contains three ATP-binding sites. There are seven homotrimeric P2X receptors (P2X1-7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5). In the lungs and airways, ATP activates P2X3 and P2X2/3 receptors (P2X3R, P2X2/3R, respectively) localized on vagal sensory nerve terminals resulting in bronchoconstriction, and cough, and probably also localized release of pro-inflammatory neuropeptides via the axon reflex. Currently, several P2X3R and P2X2/3R antagonists are being developed as drug-candidates for the treatment of chronic cough. This report presents the receptor affinity data of a novel water-soluble small molecule, DT-0111, that acts as a selective P2X3R antagonist.

Exogenous spermidine alleviates diabetic cardiomyopathy via suppressing reactive oxygen species, endoplasmic reticulum stress, and Pannexin-1-mediated ferroptosis.

Diabetic cardiomyopathy (DCM) is a serious complication and death cause of diabetes mellitus (DM). Recent cardiology studies suggest that spermidine (SPD) has cardioprotective effects. Here, we verified the hypothesis of SPD's protective effects on DCM. Therefore, db/db mice and primary neonatal mouse cardiomyocytes were used to observe the effects of SPD. Immunoblotting showed that ornithine decarboxylase (ODC) and SPD/spermine N1-acetyltransferase (SSAT) were downregulated and upregulated in the myocardium of db/db mice, respectively. We found that diabetic mice showed cardiac dysfunction in 12 weeks. Conversely, exogenous SPD could improve cardiac functions and reduce the deposition of collagens, myocardial damage, reactive oxygen species (ROS) levels, and endoplasmic reticulum stress (ERS) in diabetic mouse hearts. Our results also demonstrated that cardiomyocytes displayed ferroptosis and then activated Pannexin-1 expression, which resulted in the increase of the extracellular adenosine triphosphate (ATP). Subsequently, increased ATP as a paracrine molecule combined to purinergic receptor P2X7 to activate ERK1/2 signaling pathway in cardiomyocytes and activated NCOA4-mediated ferroptinophagy to promote lipid peroxidation and ferroptosis. Interestingly, SPD could reverse these molecular processes. Our findings indicate an important new mechanism for DCM and suggest that SPD has potential applicability to protect against deterioration of cardiac function with DCM.

Regulation of Hypoxic-Adenosinergic Signaling by Estrogen: Implications for Microvascular Injury.

Loss of estrogen, as occurs with normal aging, leads to increased inflammation, pathologic angiogenesis, impaired mitochondrial function, and microvascular disease. While the influence of estrogens on purinergic pathways is largely unknown, extracellular adenosine, generated at high levels by CD39 and CD73, is known to be anti-inflammatory in the vasculature. To further define the cellular mechanisms necessary for vascular protection, we investigated how estrogen modulates hypoxic-adenosinergic vascular signaling responses and angiogenesis. Expression of estrogen receptors, purinergic mediators inclusive of adenosine, adenosine deaminase (ADA), and ATP were measured in human endothelial cells. Standard tube formation and wound healing assays were performed to assess angiogenesis in vitro. The impacts on purinergic responses in vivo were modeled using cardiac tissue from ovariectomized mice. CD39 and estrogen receptor alpha (ERα) levels were markedly increased in presence of estradiol (E2). Suppression of ERα resulted in decreased CD39 expression. Expression of ENT1 was decreased in an ER-dependent manner. Extracellular ATP and ADA activity levels decreased following E2 exposure while levels of adenosine increased. Phosphorylation of ERK1/2 increased following E2 treatment and was attenuated by blocking adenosine receptor (AR) and ER activity. Estradiol boosted angiogenesis, while inhibition of estrogen decreased tube formation in vitro. Expression of CD39 and phospho-ERK1/2 decreased in cardiac tissues from ovariectomized mice, whereas ENT1 expression increased with expected decreases in blood adenosine levels. Estradiol-induced upregulation of CD39 substantially increases adenosine availability, while augmenting vascular protective signaling responses. Control of CD39 by ERα follows on transcriptional regulation. These data suggest novel therapeutic avenues to explore in the amelioration of post-menopausal cardiovascular disease, by modulation of adenosinergic mechanisms.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress.

Increased ATP release and its extracellular catabolism through CD73 (ecto-5'-nucleotidase) lead to the overactivation of adenosine A2A receptors (A2AR), which occurs in different brain disorders. A2AR blockade blunts mood and memory dysfunction caused by repeated stress, but it is unknown if increased ATP release coupled to CD73-mediated formation of extracellular adenosine is responsible for A2AR overactivation upon repeated stress. This was now investigated in adult rats subject to repeated stress for 14 consecutive days. Frontocortical and hippocampal synaptosomes from stressed rats displayed an increased release of ATP upon depolarization, coupled to an increased density of vesicular nucleotide transporters and of CD73. The continuous intracerebroventricular delivery of the CD73 inhibitor α,β-methylene ADP (AOPCP, 100 μM) during restraint stress attenuated mood and memory dysfunction. Slice electrophysiological recordings showed that restraint stress decreased long-term potentiation both in prefrontocortical layer II/III-layer V synapses and in hippocampal Schaffer fibers-CA1 pyramid synapses, which was prevented by AOPCP, an effect occluded by adenosine deaminase and by the A2AR antagonist SCH58261. These results indicate that increased synaptic ATP release coupled to CD73-mediated formation of extracellular adenosine contributes to mood and memory dysfunction triggered by repeated restraint stress. This prompts considering interventions decreasing ATP release and CD73 activity as novel strategies to mitigate the burden of repeated stress.

Purinergic P2X7 Receptor as a Potential Targeted Therapy for COVID-19-associated Lung Cancer Progression

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection is a serious threat to lung cancer patients. Hereby, we hypothesize that Coronavirus disease 2019 (COVID-19) may contribute to lung cancer progression by increasing extracellular adenosine triphosphate (ATP) levels and hyperactivating the purinergic P2X purinoceptor 7 receptor (P2X7R). Hyperactivation of P2X7R by increased extracellular ATP may stimulate multiple signaling pathways and factors such as NLRP3 inflammasome; as a result, interleukin (IL)-1β, and IL-18 pro-inflammatory cytokines are released, JNK, Rho kinase, HMGB1-RAGE, PI3K/AKT, hypoxia-inducible factor-1 alpha (HIF-1α), and ERK. NLRP3 activation may play a pivotal role in fatal cytokine storm in critically ill patients with COVID-19 and tumor progression in patients with lung cancer. Consequently, inhibiting these signaling pathways may deviate immune responses toward anti-tumoral responses, and suppress lung cancer progression and cytokine storms. Therefore, targeting P2X7R by means of oxidized ATP and anti-P2X7 monoclonal antibodies may provide promising therapeutic approaches to prevent lung cancer progression in COVID-19 patients; however, no clinical trials have yet been conducted, and their clinical efficacy remains to be elucidated.

Purinergic P2X7R as a potential target for pancreatic cancer.

Pancreatic cancer is one of the deadliest types of cancer, with a death rate nearly equal to the incidence. The P2X7 receptor (P2X7R) is a kind of extracellular adenosine triphosphate (ATP)-gated ion channel with special permeability, which exists in most tissues of human body and mediates inflammation-related signaling pathways and immune signal transduction after activation. P2X7R is also present on the surface of several tumor cells and is involved in tumor growth and progression. P2X7R expression in pancreatic cancer has also been identified in recent studies. Activation of P2X7R in pancreatic cancer can support the proliferation of pancreatic stellate cells, participate in protein interactions, and mediate ERK1/2, IL-6/STAT3, hCAP-18/LL-37, PI3K/AKT signaling pathways to promote pancreatic cancer progression. Inhibitors targeting P2X7R can inhibit the development of pancreatic cancer and are expected to be used in clinical therapy. Therefore, P2X7R is promising as a potential therapeutic target for pancreatic cancer. This article reviews the progress of research on P2X7R in pancreatic cancer.

Abstract PD2-07: Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer

Abstract Background: CD73 is a membrane-bound protein that by extracellular adenosine production, regulates immune cell function. Low CD73 expression is associated with better prognosis among triple-negative breast cancer (TNBC) patients. Adjuvant bisphosphonates (BPs, such as zoledronate) improve survival among post-menopausal breast cancer patients, but the mechanism for this is unknown. Since BPs also affect immune cells, we studied 1) whether zoledronate-treatment has an effect on tumor infiltrating lymphocytes (TILs) and 2) whether tumor CD73 expression affects these responses in TNBC tumors. Methods: 4T1 mouse mammary cells, stably expressing non-targeting shRNA (sh-NT, control cells) or sh-CD73 were inoculated into the mammary fat pads of Balb/c mice. The mice were treated with i.p. vehicle or zoledronate (ZOL) each 4th day for total of six times. Additionally, to deplete B-cells, the mice were treated with an anti-CD20 or control IgG antibody. Tumor sizes were assessed with a caliper, TILs were studied with immunostainings. Blood lymphocytes were analyzed with Flow cytometry. Results: Sh-CD73 cells formed significantly smaller tumors, than sh-NT cells. ZOL significantly inhibited the growth of both sh-CD73 and sh-NT tumors, but at sacrifice, this effect of ZOL was more significant against sh-CD73 tumors. ZOL increased the % of CD19+/CD21+ and CD19+/23+ in blood of both mice groups, and this effect was inhibited by anti-CD20 antibody. Although neither treatment alone had an effect, the % of CD3+/CD8+ cells in blood were increased in both mice groups by the combination of ZOL and anti-CD20-antibody. ZOL induced accumulation of B220+, CD4+ and CD8+ TILs into both tumor groups. These TIL effects were reduced with anti-CD20 antibody treatment. Anti-CD20 antibody alone inhibited significantly sh-CD73 tumor growth. Anti-CD20 antibody did not interfere with ZOL inhibition of tumor growth in either group. Conclusions: ZOL has inflammatory effects on blood lymphocytes and TILs, which may be affected by tumor expression of immune system regulating proteins, such as CD73. B cell depletion may further prevent tumor growth, in the context of CD73 inhibition. Further studies are needed to investigate whether B-cell inhibition affects ZOL-induced tumor growth inhibition. Citation Format: Nataliia Petruk, Arafat Siddiqui, Sina Tadayon, Arja Jukkola, Jorma Määttä, Pieta Mattila, Jouko Sandholm, Katri S. Selander. Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-07.

Prostaglandin D2 Controls Local Blood Flow and Sleep-Promoting Neurons in the VLPO via Astrocyte-Derived Adenosine.

Prostaglandin D2 (PGD2) is one of the most potent endogenous sleep-promoting molecules. However, the cellular and molecular mechanisms of the PGD2-induced activation of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO), the major nonrapid eye movement (NREM)-sleep center, still remains unclear. We here show that PGD2 receptors (DP1) are not only expressed in the leptomeninges but also in astrocytes from the VLPO. We further demonstrate, by performing real-time measurements of extracellular adenosine using purine enzymatic biosensors in the VLPO, that PGD2 application causes a 40% increase in adenosine level, via an astroglial release. Measurements of vasodilatory responses and electrophysiological recordings finally reveal that, in response to PGD2 application, adenosine release induces an A2AR-mediated dilatation of blood vessels and activation of VLPO sleep-promoting neurons. Altogether, our results unravel the PGD2 signaling pathway in the VLPO, controlling local blood flow and sleep-promoting neurons, via astrocyte-derived adenosine.

The adenosinergic machinery in cancer: In-tandem insights from basic mechanisms to therapy.

Extracellular adenosine (eADO) signaling has emerged as an increasingly important regulator of immune responses, including tumor immunity. eADO is mainly produced from extracellular ATP (eATP) hydrolysis. eATP is rapidly accumulated in the extracellular space following cell death or cellular stress triggered by hypoxia, nutrient starvation, or inflammation. eATP plays a pro-inflammatory role by binding and activating the P2 purinergic receptors (P2X and P2Y), while eADO has been reported in many studies to mediate immunosuppression by activating the P1 purinergic receptors (A1, A2A, A2B, and A3) in diverse immune cells. Consequently, the hydrolysis of eATP to eADO alters the immunosurveillance in the tumor microenvironment (TME) not only by reducing eATP levels but also by enhancing adenosine receptor signaling. The effects of both P1 and P2 purinergic receptors are not restricted to immune cells. Here we review the most up-to-date understanding of the tumor adenosinergic system in all cell types, including immune cells, tumor cells, and stromal cells in TME. The potential novel directions of future adenosinergic therapies in immuno-oncology will be discussed.

Adenosinergic Pathway in Parkinson's Disease: Recent Advances and Therapeutic Perspective.

Parkinson's disease (PD) is a neurodegenerative disease characterized pathologically by α-synuclein (α-syn) aggregation. In PD, the current mainstay of symptomatic treatment is levodopa (L-DOPA)-based dopamine (DA) replacement therapy. However, the development of dyskinesia and/or motor fluctuations which is relevant to levodopa is restricting its long-term utility. Given that the ability of which is to modulate the striato-thalamo-cortical loops and function to modulate basal ganglia output, the adenosinergic pathway (AP) is qualified as a potential promising non-DA target. As an indispensable component of energy production pathways, AP modulates cellular metabolism and gene regulation in both neurons and neuroglia cells through the recognition and degradation of extracellular adenosine. In addition, AP is geared to the initiation, evolution, and resolution of inflammation as well. Besides the above-mentioned crosstalk between the adenosine and dopamine signaling pathways, the functions of adenosine receptors (A1R, A2AR, A2BR, and A3R) and metabolism enzymes in modulating PD pathological process have been extensively investigated in recent decades. Here we reviewed the emerging findings focused on the function of adenosine receptors, adenosine formation, and metabolism in the brain and discussed its potential roles in PD pathological process. We also recapitulated clinical studies and the preclinical evidence for the medical strategies targeting the Ado signaling pathway to improve motor dysfunction and alleviate pathogenic process in PD. We hope that further clinical studies should consider this pathway in their monotherapy and combination therapy, which would open new vistas to more targeted therapeutic approaches.

Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients.

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A2A receptors (A2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Adenosine-independent regulation of the sleep–wake cycle by astrocyte activity

Astrocytes play a crucial role in regulating sleep–wake behavior, and adenosine signaling is generally thought to be involved. Here we show multiple lines of evidence supporting that modulation of the sleep–wake behavior by astrocyte Ca2+ activity could occur without adenosine signaling. In the basal forebrain and the brainstem, two brain regions that are known to be essential for sleep–wake regulation, chemogenetically-induced astrocyte Ca2+ elevation significantly modulated the sleep–wake cycle. Although astrocyte Ca2+ level positively correlated with the amount of extracellular adenosine, as revealed by a genetically encoded adenosine sensor, we found no detectable change in adenosine level after suppressing astrocyte Ca2+ elevation, and transgenic mice lacking one of the major extracellular ATP-adenosine conversion enzymes showed similar extracellular adenosine level and astrocyte Ca2+-induced sleep modulation. Furthermore, astrocyte Ca2+ is dependent primarily on local neuronal activity, causing brain region-specific regulation of the sleep–wake cycle. Thus, neural activity-dependent astrocyte activity could regulate the sleep–wake behavior independent of adenosine signaling.

Purinergic signaling: A gatekeeper of blood-brain barrier permeation.

This review outlined evidence that purinergic signaling is involved in the modulation of blood-brain barrier (BBB) permeability. The functional and structural integrity of the BBB is critical for maintaining the homeostasis of the brain microenvironment. BBB integrity is maintained primarily by endothelial cells and basement membrane but also be regulated by pericytes, neurons, astrocytes, microglia and oligodendrocytes. In this review, we summarized the purinergic receptors and nucleotidases expressed on BBB cells and focused on the regulation of BBB permeability by purinergic signaling. The permeability of BBB is regulated by a series of purinergic receptors classified as P2Y1, P2Y4, P2Y12, P2X4, P2X7, A1, A2A, A2B, and A3, which serve as targets for endogenous ATP, ADP, or adenosine. P2Y1 and P2Y4 antagonists could attenuate BBB damage. In contrast, P2Y12-mediated chemotaxis of microglial cell processes is necessary for rapid closure of the BBB after BBB breakdown. Antagonists of P2X4 and P2X7 inhibit the activation of these receptors, reduce the release of interleukin-1 beta (IL-1β), and promote the function of BBB closure. In addition, the CD39/CD73 nucleotidase axis participates in extracellular adenosine metabolism and promotes BBB permeability through A1 and A2A on BBB cells. Furthermore, A2B and A3 receptor agonists protect BBB integrity. Thus, the regulation of the BBB by purinergic signaling is complex and affects the opening and closing of the BBB through different pathways. Appropriate selective agonists/antagonists of purinergic receptors and corresponding enzyme inhibitors could modulate the permeability of the BBB, effectively delivering therapeutic drugs/cells to the central nervous system (CNS) or limiting the entry of inflammatory immune cells into the brain and re-establishing CNS homeostasis.

The ectonucleotidases CD39 and CD73 on T cells: The new pillar of hematological malignancy.

Hematological malignancy develops and applies various mechanisms to induce immune escape, in part through an immunosuppressive microenvironment. Adenosine is an immunosuppressive metabolite produced at high levels within the tumor microenvironment (TME). Adenosine signaling through the A2A receptor expressed on immune cells, such as T cells, potently dampens immune responses. Extracellular adenosine generated by ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) molecules is a newly recognized 'immune checkpoint mediator' and leads to the identification of immunosuppressive adenosine as an essential regulator in hematological malignancies. In this Review, we provide an overview of the detailed distribution and function of CD39 and CD73 ectoenzymes in the TME and the effects of CD39 and CD73 inhibition on preclinical hematological malignancy data, which provides insights into the potential clinical applications for immunotherapy.

Aberrant Adenosine Triphosphate Release and Impairment of P2Y2-Mediated Signaling in Sarcoglycanopathies

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune–mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune–mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.

Single-Cell Phenotyping of CD73 Expression Reveals the Diversity of the Tumor Immune Microenvironment and Reflects the Prognosis of Bladder Cancer

The cutting edge of cancer immunotherapy extends to ecto-5′-nucleotidase (CD73), a cell membrane enzyme that targets the metabolism of extracellular adenosine. We herein focused on the expression of CD73 to clarify the state of CD73 positivity in cancer immunity and tumor microenvironment, thereby revealing a new survival predictor for patients with bladder cancer (BCa). We used clinical tissue microarrays of human BCa and simultaneously performed the fluorescent staining of cell type-specific markers (CD3, CD8, Foxp3, programmed cell death protein 1, and programmed death-ligand 1 [PD-L1]) and CD73 together with DAPI for nuclear staining. In total, 156 participants were included. Multiplexed cellular imaging revealed a unique interaction between CD73 expression and CD8+ cytotoxic T cells (CTLs) and Foxp3+ regulatory T (Treg) cells in human BCa, showing the high infiltration of CD8+CD73+ CTLs and Foxp3+CD73+ Treg cells in tumors to be associated with tumorigenesis and poor prognosis in BCa. Interestingly, from a biomarker perspective, the high infiltration of CD73+ Treg cells in tumors was identified as an independent risk factor for overall survival in addition to clinicopathologic features. Regarding the relationship between immune checkpoint molecules and CD73 expression, both CD73+ CTLs and CD73+ Treg cells tended to coexpress programmed cell death protein 1 as tumor invasiveness and nuclear grade increased. Additionally, they may occupy a spatial niche located distantly from PD-L1+ cells in tumors to interfere less with the cancerous effects of PD-L1+ cells. In conclusion, the present results on the status of CD73 in cancer immunity suggest that CD73 expression on specific T-cell types has a negative immunoregulatory function. These findings may provide further insights into the immunobiological landscape of BCa, which may be translationally linked to improvements in future immunotherapy practice.