The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Preclinical and clinical aspects of P2X receptors as a common route in different diseases: A meeting report.

PRESTO was established in 2022 and is a concerted effort by leading European experts in the field of P2XRs and extracellular ATP to promote and advance the transition to the clinic of P2XR-targeting therapies. Following the inaugural meeting in Ferrara which set the foundations of the action and generated interest from many groups and institutes, the second meeting covered the preclinical and clinical aspects of P2XRs as a common route in different diseases, recognising the multidisciplinary and collaborative approach required for a number of medical conditions.

Bitter Taste Receptor Agonist Denatonium Inhibits Stemness Characteristics in Hematopoietic Stem/Progenitor Cells.

Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.

The P2X7 Receptor in Oncogenesis and Metastatic Dissemination: New Insights on Vesicular Release and Adenosinergic Crosstalk.

The tumor niche is an environment rich in extracellular ATP (eATP) where purinergic receptors have essential roles in different cell subtypes, including cancer, immune, and stromal cells. Here, we give an overview of recent discoveries regarding the role of probably the best-characterized purinergic receptor in the tumor microenvironment: P2X7. We cover the activities of the P2X7 receptor and its human splice variants in solid and liquid cancer proliferation, dissemination, and crosstalk with immune and endothelial cells. Particular attention is paid to the P2X7-dependent release of microvesicles and exosomes, their content, including ATP and miRNAs, and, in general, P2X7-activated mechanisms favoring metastatic spread and niche conditioning. Moreover, the emerging role of P2X7 in influencing the adenosinergic axis, formed by the ectonucleotidases CD39 and CD73 and the adenosine receptor A2A in cancer, is analyzed. Finally, we cover how antitumor therapy responses can be influenced by or can change P2X7 expression and function. This converging evidence suggests that P2X7 is an attractive therapeutic target for oncological conditions.

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.

Emerging roles of purinergic signaling in anti-cancer therapy resistance.

Cancer is a complex disease with a rapid growing incidence and often characterized by a poor prognosis. Although impressive advances have been made in cancer treatments, resistance to therapy remains a critical obstacle for the improvement of patients outcome. Current treatment approaches as chemo-, radio-, and immuno-therapy deeply affect the tumor microenvironment (TME), inducing an extensive selective pressure on cancer cells through the activation of the immune system, the induction of cell death and the release of inflammatory and damage-associated molecular patterns (DAMPS), including nucleosides (adenosine) and nucleotides (ATP and ADP). To survive in this hostile environment, resistant cells engage a variety of mitigation pathways related to metabolism, DNA repair, stemness, inflammation and resistance to apoptosis. In this context, purinergic signaling exerts a pivotal role being involved in mitochondrial function, stemness, inflammation and cancer development. The activity of ATP and adenosine released in the TME depend upon the repertoire of purinergic P2 and adenosine receptors engaged, as well as, by the expression of ectonucleotidases (CD39 and CD73) on tumor, immune and stromal cells. Besides its well established role in the pathogenesis of several tumors and in host-tumor interaction, purinergic signaling has been recently shown to be profoundly involved in the development of therapy resistance. In this review we summarize the current advances on the role of purinergic signaling in response and resistance to anti-cancer therapies, also describing the translational applications of combining conventional anticancer interventions with therapies targeting purinergic signaling.

P2X7 receptor isoform B is a key drug resistance mediator for neuroblastoma.

Drug resistance is a major challenge for all oncological treatments that involve the use of cytotoxic agents. Recent therapeutic alternatives cannot circumvent the ability of cancer cells to adapt or alter the natural selection of resistant cells, so the problem persists. In neuroblastoma, recurrence can occur in up to 50% of high-risk patients. Therefore, the identification of novel therapeutic targets capable of modulating survival or death following classical antitumor interventions is crucial to address this problem. In this study, we investigated the role of the P2X7 receptor in chemoresistance. Here, we elucidated the contributions of P2X7 receptor A and B isoforms to neuroblastoma chemoresistance, demonstrating that the B isoform favors resistance through a combination of mechanisms involving drug efflux via MRP-type transporters, resistance to retinoids, retaining cells in a stem-like phenotype, suppression of autophagy, and EMT induction, while the A isoform has opposite and complementary roles.

Administration of P2X7 Receptor Blockers in Oncological Experimental Models.

The tumor microenvironment is rich in components that strongly influence cancer cell survival. One of the pivotal molecules present at the tumor bed is ATP, which has an essential role in promoting cancer proliferation and metastasis and immune responses via its receptor P2X7. Several studies have proved the efficacy of P2X7 pharmacological blockade in inhibiting primary and metastatic tumor growth in preclinical models. Here we describe the experimental procedures that we optimized to test P2X7 roles in carcinogenesis by antagonist administration. Special attention is paid to their concentrations and routes of administration. The depicted in vitro models include cell count and viability assays, which are useful to test P2X7 roles in cell proliferation and vitality, and the soft agar colony formation test that allows investigation of the transforming and invading abilities of tumor cells. We also describe systemic and intramass administration of P2X7 blockers in murine models of melanoma and leukemia. Both xenotransplant and syngeneic experimental tumor models are detailed.

A2A Receptor Contributes to Tumor Progression in P2X7 Null Mice.

ATP and adenosine are key constituents of the tumor niche where they exert opposite and complementary roles. ATP can be released in response to cell damage or actively released by tumor cells and subsequently degraded into adenosine, which accumulates within the tumor microenvironment. Notably, while ATP promotes immune eradicating responses mainly via the P2X7 receptor (P2X7R), extracellular adenosine acts as a potent immune suppressor and facilitates neovascularization thanks to the A2A receptor (A2AR). To date, studies exploring the interplay between P2X7R and A2AR in the tumor microenvironment are as yet missing. Here, we show that, in C57/bl6 P2X7 null mice inoculated with B16-F10 melanoma cells, several pro-inflammatory cytokines, including interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 17 (IL-17), interferon gamma (IFN-γ) were significantly decreased, while the immune suppressant transforming growth factor beta (TGF-ß) was almost three-fold increased. Interestingly, tumors growing in P2X7-null mice upregulated tumor-associated and splenic A2AR, suggesting that immunosuppression linked to lack of the P2X7R might depend upon A2AR overexpression. Immunohistochemical analysis showed that tumor cells' A2AR expression was increased, especially around necrotic areas, and that vascular endothelial growth factor (VEGF) and the endothelial marker CD31 were upregulated. A2AR antagonist SCH58261 treatment reduced tumor growth similarly in the P2X7 wild type or null mice strain. However, SCH58261 reduced VEGF only in the P2X7 knock out mice, thus supporting the hypothesis of an A2AR-mediated increase in vascularization observed in the P2X7-null host. SCH58261 administration also significantly reduced intratumor TGF-ß levels, thus supporting a key immune suppressive role of A2AR in our model. Altogether, these results indicate that in the absence of host P2X7R, the A2AR favors tumor growth via immune suppression and neovascularization. This study shows a novel direct correlation between P2X7R and A2AR in oncogenesis and paves the way for new combined therapies promoting anti-cancer immune responses and reducing tumor vascularization.

Irradiation causes senescence, ATP release, and P2X7 receptor isoform switch in glioblastoma.

Glioblastoma (GBM) is the most lethal brain tumor in adults. Radiation, together with temozolomide is the standard treatment, but nevertheless, relapse occurs in nearly all cases. Understanding the mechanisms underlying radiation resistance may help to find more effective therapies. After radiation treatment, ATP is released into the tumor microenvironment where it binds and activates purinergic P2 receptors, mainly of the P2X7 subtype. Two main P2X7 splice variants, P2X7A and P2X7B, are expressed in most cell types, where they associate with distinct biochemical and functional responses. GBM cells widely differ for the level of P2X7 isoform expression and accordingly for sensitivity to stimulation with extracellular ATP (eATP). Irradiation causes a dramatic shift in P2X7 isoform expression, with the P2X7A isoform being down- and the P2X7B isoform up-modulated, as well as extensive cell death and overexpression of stemness and senescence markers. Treatment with P2X7 blockers during the post-irradiation recovery potentiated irradiation-dependent cytotoxicity, suggesting that P2X7B activation by eATP generated a trophic/growth-promoting stimulus. Altogether, these data show that P2X7A and B receptor isoform levels are inversely modulated during the post-irradiation recovery phase in GBM cells.

Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation.

Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME.

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells.

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

Cancer Metabostemness and Metabolic Reprogramming via P2X7 Receptor.

The heterogeneity of tumor cell mass and the plasticity of cancer cell phenotypes in solid tumors allow for the insurgence of resistant and metastatic cells, responsible for cancer patients' clinical management's main challenges. Among several factors that are responsible for increased cancer aggression, metabolic reprogramming is recently emerging as an ultimate cancer hallmark, as it is central for cancer cell survival and self-renewal, metastasis and chemoresistance. The P2X7 receptor, whose expression is upregulated in many solid and hematological malignancies, is also emerging as a good candidate in cancer metabolic reprogramming and the regulation of stem cell proliferation and differentiation. Metabostemness refers to the metabolic reprogramming of cancer cells toward less differentiated (CSCs) cellular states, and we believe that there is a strong correlation between metabostemness and P2X7 receptor functions in oncogenic processes. Here, we summarize important aspects of P2X7 receptor functions in normal and tumor tissues as well as essential aspects of its structure, regulation, pharmacology and its clinical use. Finally, we review current knowledge implicating P2X7 receptor functions in cancer-related molecular pathways, in metabolic reprogramming and in metabostemness.

Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein.

Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface.

Corrigendum: Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells.

[This corrects the article DOI: 10.3389/fonc.2020.01225.].

Role of ATP in Extracellular Vesicle Biogenesis and Dynamics.

Adenosine triphosphate (ATP) is among the molecules involved in the immune response. It acts as danger signal that promotes inflammation by activating both P2X and P2Y purinergic receptors expressed in immune cells, including microglia, and tumor cells. One of the most important receptors implicated in ATP-induced inflammation is P2X7 receptor (P2X7R). The stimulation of P2X7R by high concentration of ATP results in cell proliferation, inflammasome activation and shedding of extracellular vesicles (EVs). EVs are membrane structures released by all cells, which contain a selection of donor cell components, including proteins, lipids, RNA and ATP itself, and are able to transfer these molecules to target cells. ATP stimulation not only promotes EV production from microglia but also influences EV composition and signaling to the environment. In the present review, we will discuss the current knowledge on the role of ATP in the biogenesis and dynamics of EVs, which exert important functions in physiology and pathophysiology.

P2X7 Receptor in Hematological Malignancies.

The P2X7 receptor is an ion channel gated by the nucleotide ATP, known for its role in immune responses and recently emerging as a critical onco-promoting factor. Lymphocytes, myeloid cells, and their precursors were among the first cells proved to express a functional P2X7 receptor; therefore, it is not surprising that lymphoproliferative and myeloproliferative diseases, also known as hematological malignancies, were shown to be related in their insurgence and progression to P2X7 alterations. Here, we overview established and recent literature relating P2X7 with the biological mechanisms underlying leukemias, lymphomas, and multiple myeloma development. Particular attention is paid to studies published in the very recent past correlating P2X7 with ATP concentration in the leukemic microenvironment and P2X7 overexpression to acute myeloid leukemia aggressiveness and response to chemotherapy. The described literature strongly suggests that P2X7 and its genetic variants could be regarded as potential new biomarkers in hematological malignancies and that both P2X7 antagonists and agonists could emerge as new therapeutic tools alone or in combination with traditional chemotherapy.