Lucas Nussbaum
email: lucas.nussbaum@imag.fr

Olivier Richard
email: olivier.richard@imag.fr

Lightweight Emulation to Study Peer-to-Peer Systems *

The current methods used to test and study peer-topeer systems (namely modeling, simulation, or execution on real testbeds) often show limits regarding scalability, realism and accuracy. This paper describes and evaluates P2PLab, our framework to study peer-to-peer systems by combining emulation (use of the real studied application within a configured synthetic environment) and virtualization. P2PLab is scalable (it uses a distributed network model) and has good virtualization characteristics (many virtual nodes can be executed on the same physical node by using process-level virtualization). Experiments with the BitTorrent file-sharing system complete this paper and demonstrate the usefulness of this platform.

Introduction

Peer-to-peer systems have become more and more popular over the last few years, and this popularity often required changes that made them more and more complex. Due to this ever increasing complexity, the development and the study of peer-to-peer systems have become more difficult : we need ways to ensure that a peer-to-peer application will work properly on thousands of nodes, or ways to understand applications running on thousands of nodes.

Distributed applications are traditionally studied using mathematical modeling, simulation, and execution on a real system. Simulation consists in using a model of the application's code in a synthetic environment. This method is widely used, and gives valuable results easily. However, it is often difficult to simulate efficiently a large number of nodes using a complex model : a trade-off between the realism of the model and the number of nodes always has to be made.

On the other side, one can run the real application to study on a real-world experimentation platform like PlanetLab [START_REF] Chun | Planet-Lab: An Overlay Testbed for Broad-Coverage Services[END_REF]. But the environment is then difficult to control and modify (since it depends heavily on the real system itself), and results are often difficult to reproduce (since the environmental conditions may vary a lot between experiments). This kind of real-world experiments is needed when developing a peer-to-peer system, but it doesn't satisfy all needs.

Between those two approaches, this paper explores an intermediate solution using emulation and virtualization, and shows that such an approach can provide interesting results when used to study peer-to-peer systems.

Emulation and Virtualization have to be distinguished :

Emulation consists in providing a modified environment to the studied application, to match the conditions of the experiment. Determining which resources to emulate (and with which precision) has to be considered as a trade-off between realism and cost (a precise emulation of conditions can be very CPU-intensive). For example, when studying peer-to-peer systems, network emulation is important, while emulation of different types of hard disks is probably not necessary. Emulation is often costly, and its cost is often difficult to evaluate, because it depends both on the quality of emulation and on the emulation parameters : emulating an high-latency network will be more expensive than emulating a low-latency one.

Virtualization of resources allows to share a resource between several instances of an application. It is required to be able to study a large number of co-existing nodes. In the field of distributed systems, virtualization allows to execute several instances of the application or the operating system on the same physical machine, thus increasing the number of nodes available for the experiment. Of course, since the resources of the physical machine are shared between instances, the fairness of this share is an important issue. Like the precision of emulation, the quality of the fairness is a trade-off.

Related Works

A lot of work occurred recently in the virtualization area, with different approaches. Linux Vserver [START_REF] Project | [END_REF] is a patch for the Linux kernel adding contexts and managing interactions between them, allowing several environments to share the same kernel with a very low overhead. User Mode Linux [START_REF] Dike | A user-mode port of the Linux kernel[END_REF] is a port of the Linux kernel to a Linux process. And Xen [START_REF] Barham | Xen and the art of virtualization[END_REF] uses paravirtualization to give the ability to run simultaneously several operating systems. Those operating systems have to be modified to run over the Xen microkernel, which is in charge of sharing resources and providing virtual devices.

Regarding network emulation (by far the most important resource to control while studying peer-to-peer systems), there are both low-level tools which work at the packet level to emulate different network connections (varying bandwidth and latency) and higher level tools which allow to build a complex synthetic network topology.

The low level tools include Dummynet [START_REF] Rizzo | Dummynet: a simple approach to the evaluation of network protocols[END_REF], which runs on FreeBSD and is the most popular network emulator, NISTNet [START_REF] Carson | NIST Net: a Linux-based network emulation tool[END_REF] (which runs on Linux 2.4), and Linux 2.6's Traffic Control (TC) and iproute2 tools [START_REF]Routing and Traffic[END_REF] (using NetEm [START_REF] Hemminger | Network emulation with NetEem[END_REF]). Those tools schedule inbound and/or outbound packets to control bandwidth and delay, and emulate network problems such as packet loss.

Higher level tools use the former low-level tools to emulate complex topologies. NetBed [START_REF] White | An integrated experimental environment for distributed systems and networks[END_REF] combines real nodes using RTC or DSL lines, nodes using Dummynet, and simulated nodes (using Network Simulator Emulation Layer) to provide experimental environments. Modelnet [START_REF] Vahdat | Scalability and accuracy in a large-scale network emulator[END_REF] uses cluster nodes split in two groups : the application under study runs on edge nodes while the Modelnet core nodes use Dummynet to emulate a network topology. Modelnet uses a distillation phase to make a trade-off between accuracy and scalability to be able to emulate the network on a small number of nodes.

The current virtualization tools target high realism and have a relatively poor virtualization ratio (number of virtual nodes / number of physical nodes). One of the problems is that they virtualize a full operating system (kernel, libraries, application). This is often not needed for peer-to-peer applications, since they are relatively well self-contained.

Another issue is network emulation : current tools target a realistic emulation of the core network (congestion, routing, etc inside the core links). Most peerto-peer applications are used on the edge nodes of the Internet, often on home computer with DSL connections. Therefore, even if some aspects of the Internet core are important (e.g latency, for experiments involving locality), the emphasis can be put on the emulation of the link between the edge nodes and their Internet Service Provider. This link is the bottleneck in most (if not all) cases.

P2PLab

Overview

P2PLab is our emulation tool for studying peer-topeer systems. It targets high efficiency (large number of virtual nodes can be studied on a low number of physical nodes), and scalability (experiments can be done with thousands of nodes).

P2PLab virtualizes at the process level, not at the system level, to provide better scalability. It runs on FreeBSD, since it uses Dummynet for network emulation. A decentralized approach is used to emulate network topologies, allowing better scalability.

First, we will verify that FreeBSD is a suitable platform for P2PLab by checking that its scheduler is scalable and provides a good level of fairness. Then, P2PLab's virtualization system will be described.

In a second part, the network emulation model of P2PLab will be presented.

Virtualization

While most virtualization systems virtualize on the operating system level, it is not mandatory here since the goal is to study peer-to-peer systems. It was therefore decided to virtualize the process' network identity by binding each process to its own IP address. The FreeBSD operating system was chosen for P2PLab because of the availability of Dummynet [START_REF] Rizzo | Dummynet: a simple approach to the evaluation of network protocols[END_REF], FreeBSD's network traffic shaper. But it was still necessary to test whether FreeBSD was a suitable platform to run a very large number of processes without compromising our experiment's results. FreeBSD has two schedulers : the classic 4BSD scheduler, and the more modern ULE scheduler (which is similar to Linux 2.6's scheduler). It was decided to evaluate both. The evaluation took place on nodes of the GridExplorer system, part of the French grid research project Grid5000 [START_REF] Cappello | Grid'5000: a large scale, reconfigurable, controlable and monitorable Grid platform[END_REF]. The nodes are Dual-Opteron 2Ghz with 2 Gb of RAM and gigabit ethernet. The first experiment was to evaluate the scheduler's ability to run multiple processes by running a large number of concurrent non-memory-intensive processes (each process, when started alone, needed about half a second of CPU time to complete). Figure 1 shows that total calculation time scales linearly with both the 4BSD and the ULE scheduler. However, the overhead is higher with the ULE scheduler.

Then, the same experiment was done with memoryintensive processes. Figure 2 shows clearly that results shouldn't be trusted as soon as swap is used (the figure only shows results with the 4BSD scheduler. Results with the ULE scheduler were similar).

Fairness between processes is also important. To measure it, concurrent instances of a CPU-intensive process were started every second, and the real run time of each instance was measured. Figure 3 shows that there are serious fairness problems in the ULE scheduler (some processes seem to run alone on the second CPU !). Fairness of the 4BSD and Linux 2.6 schedulers were better but not perfect. This kind of process schedulers try to maximize throughput and interactivity, and fairness is not a priority. Some different settings were tried for the timer interrupt frequency (HZ) and the quantum (time allocated to a process before switching to another one), but it didn't improve the results significantly. After those first experiments, the 4BSD scheduler chosen for P2PLab. As said earlier, virtualization is made at the level of the process' network identity : instances on the same physical system share all resources (filesystem, memory etc.) as normal processes do. However, each process has its own IP address on the network. The main IP address of each physical system is kept for administration purposes. The IP addresses of the virtual nodes are configured as interface aliases as shown in figure 4 (most UNIX systems, including Linux and FreeBSD, allow each network interface to be assigned several IP addresses through an aliasing system). Evaluation showed that interface aliases produced no overhead compared to the normal assignment of an IP address to an interface.

To avoid namespace conflicts, the adresses of the virtual nodes were chosen in different subnet. Figure 4 shows an example configuration using the 192.168.38/24 network for administration and the 10.0.0.0/8 network for virtual nodes. To bind an application to a particular IP address, we chose to intercept some network system calls. Several options are available : modifying the application under study, linking the application with a specific library, using ptrace() to intercept system calls, modifying the kernel, or modifying the C library.

In P2PLab, we chose to modify the FreeBSD C library, which provided a good compromise between complexity and efficiency. We modified the bind(), connect() and listen() library calls using a naive approach :

• When bind() is called, the my addr parameter is modified to restrict the bind to the IP address specified by the BINDIP environment variable.

• When connect() or listen() are called, bind() is called before to restrict connect() or listen() to the IP address in BINDIP. If another bind() was made before, this one will fail, but we ignore the error in this case.

This approach doubles the number of system calls for connect() and listen() and only works for TCP connections. A similar approach is possible for UDP.

Tests showed that this libc modification was working as expected with programs in Perl, Python, Ruby, TCL, C, Java (with Sun's JDK). The only case where this approach failed to work was statically compiled C programs.

Then, we evaluated the overhead of this approach during the establishment of TCP connections. The test program was connecting to a local server and disconnecting as soon as the connection was established. We measured that the duration of a connection/disconnection cycle was 10.22µs without the modification, to compare to 10.79µs with the modification : the cost of this approach is very low, even in the worst case.

Network Emulation

Current network topology emulators like Model-Net [START_REF] Vahdat | Scalability and accuracy in a large-scale network emulator[END_REF] target a realistic emulation of the core network. But most peer-to-peer applications run on nodes on the edge of the Internet : while the traffic in the core of the Internet can influence the peer-to-peer system behaviour (congestion between providers can increase latency, for example), the main bottleneck for end nodes is often the link between the user and its Internet service provider (ISP). Therefore, it is possible to model the Internet by reproducing what the end node really sees, excluding what is less important from the end node point of view.

P2PLab's emulation model allows to control :

• bandwidth, latency and packet loss rate on the network link between the end node and its ISP ;

• latency between group of nodes, allowing to study problems involving locality. Figure 5 shows an example topology which we successfully emulated using P2PLab. Figure 6 presents some ping results between nodes of this topology (there was no other traffic between the virtual nodes when the pings were made). Let's use the measurement between 10.1.3.207 and 10.2.2.117 as an example. The latency of 853 ms can be decomposed in :

• 20 ms of delay when the packet goes out of 10.1. Network emulation is achieved in a decentralized way : each physical node is in charge of the network emulation for its virtual nodes. On each node, emulation is done with Dummynet [START_REF] Rizzo | Dummynet: a simple approach to the evaluation of network protocols[END_REF], the FreeBSD traffic shaper integrated into FreeBSD's firewall (IPFW). Both incoming and outgoing packets are delayed by Dummynet. Several firewall rules are needed for each virtual node. For example, the physical node hosting 10.2.3.207 in the topology described in figure 5 will need :

• One rule for incoming packets for each virtual node hosted on this node ;

• One rule for outgoing packets for each virtual node hosted on this node ;

• One rule for each link between network groups, to delay packets going from network groups hosted on this system to other network groups. The overhead caused by Dummynet was evaluated by building a token ring on a single machine. The network of each virtual machine is emulated, causing two Dummynet rules to be added per virtual node. Figure 7 shows that the overhead caused by selecting the Dummynet rule to apply is important, because FreeBSD's IPFW examines firewall rules linearly. However, for a relatively low number of virtual nodes, the overhead is still reasonable (3.5 ms for 200 virtual nodes per physical node) compared to the latency of the networks we are going to emulate.

Study of BitTorrent with P2PLab

In this section, we confront our emulation platform with BitTorrent, showing that P2PLab is a suitable ex-perimentation platform to study complex peer-to-peer system.

Introduction

BitTorrent [START_REF] Cohen | Incentives build robustness in BitTorrent[END_REF] is a popular peer-to-peer file distribution system. It provides very good performance by ensuring that downloaders cooperate by sharing parts they have already downloaded through a complex reciprocation system. It has already been largely evaluated through analysis of large scale utilization [START_REF] Pouwelse | The Bittorrent P2P file-sharing system: Measurements and analysis[END_REF][START_REF] Izal | Dissecting BitTorrent: five months in a torrent's lifetime[END_REF], analytical modeling [START_REF] Qiu | Modeling and performance analysis of BitTorrent-like peer-to-peer networks[END_REF] or simulation [START_REF] Bharambe | Analyzing and improving BitTorrent performance[END_REF].

However, those works have never been compared to large scale studies on real world systems, or to studies using emulation. BitTorrent is an engineering work, not a research prototype, and several parts of its code are very complex. The large number of constants used as parameters of all the important algorithms makes it very hard to model accurately.

A BitTorrent 4.0.4 client was used for all experiments. It was slightly modified to allow data collection (a timestamp was added to the default output). The experiments took place on the GridExplorer system, already described in section 3.2 on page 2.

P2PLab Folding Ratio

P2PLab targets an high virtualization ratio : it should be possible to run many virtual nodes on the same physical system, thus allowing to do experiments with a very large number of nodes. But the concurrent execution of several instances of the application must not affect the results.

The first experiment compares the download of a 16 MB file by 160 clients. The file size is not important in BitTorrent, since the file is always divided in pieces of 256 KB. The file is provided by 4 seeders. All nodes (both downloaders and seeders) have a network connection with a download rate of 2 mbps, an upload rate of 128 kbps, and a latency of 30 ms, reproducing the conditions of a DSL connection. The clients are started with a 10 s interval. When the clients have finished the download of the file, they stay online and become seeders, continuing to upload data to the downloaders.

The 160 clients are deployed successively on 160 physical nodes, 16 physical nodes (10 virtual nodes per physical node), 8, 4 and 2 physical nodes.

Figure 8 shows the evolution of the download of all 160 clients when they are deployed on 160 physical nodes. One can see that with those parameters, all the cases of a BitTorrent download are represented :

• First (short) part of the download when only initial seeders are able to upload data ; • Second part when all downloaders start contributing to each other ;

• Third part when the first downloaders become seeders and help other peers finishing their download faster.

Therefore, those parameters are a realistic workload to test P2PLab's virtualization capabilities. Figure 9 shows that the experiment took place without any overhead, even with 80 virtual nodes on each physical node : results are nearly identical. The potential sources of overhead were investigated, and it was determined that the first limiting factor was the network speed : with other (slightly faster) emulated network settings, the platform's gigabit network was saturated by the downloads.

Other overhead sources were considered : during the experiment, we monitored the system load, the memory usage, and the disk I/O on every physical node. None of them was a problem during our experiments.

P2PLab Scalability

P2PLab targets high scalability : it should be possible to run experiments with a large number of nodes. We tried to study the download of a 16 MB file by 5754 clients, under the same network conditions as in the first experiment. The 5760 virtual nodes (5754 clients, 4 seeders, one tracker) are hosted on 180 physical nodes (32 virtual nodes per physical node). The clients are started every 0.25 s so the entire experiment took less than one hour. The experiment succeeded, and figure 10 shows the evolution of the download on 115 selected clients (every 50 clients, so clients numbered 0, 50, 100, 150, ..., 5750) 1 . One can see that most clients finish their downloads nearly at the same time, at the end of the experiment. This is confirmed by figure 11, which shows the number of clients having completed their download over time.

Future Work

Our decentralized network model doesn't consider the problem of congestion in the core links (between internet service providers). Its role in the performance of peer-to-peer systems need to be determined through experiments on PlanetLab [START_REF] Chun | Planet-Lab: An Overlay Testbed for Broad-Coverage Services[END_REF] or DSL-Lab [START_REF]DSL-Lab[END_REF]. Then, we will be able to modify our network model to include it.

Another issue left open is the handling of shared resources. We have shown that one of the most important, CPU time, wasn't shared equally between processes. While the use of off-the-shelf software for emulation makes it difficult to provide fair-sharing, it would be interesting to improve the situation, or at least to provide it for some resources. Developing a more deterministic process scheduler would already allow solve the problem of the scheduler's fairness.

The use of FreeBSD for P2PLab wasn't easy : the dominant operating system on Grid5000 [START_REF] Cappello | Grid'5000: a large scale, reconfigurable, controlable and monitorable Grid platform[END_REF] (the experimentation platform we used) is Linux, and using FreeBSD instead required quite a lot of changes. After evaluating (and maybe improving) the comparable tools on Linux, we will consider a switch to Linux as the basis for P2PLab.

Another problem with FreeBSD is the way its firewall evaluates rules : as shown in section 3.3, the rules are evaluated linearly, which is clearly not optimal in the case of P2PLab, where we want to choose only one rule inside a large number of them. Implementing an hash-based mechanism for rules lookup would be much more efficient.

The experiments made with P2PLab on BitTorrent are using sensible but unrealistic parameters. Coupling P2PLab with a realistic input generator would allow to easily generate realistic environments and behaviours. Finally, we need to validate P2PLab against other peer-to-peer systems, to verify that it allows to answer a wide range of questions on a wide range of systems.

Summary and Conclusion

With the increase of the resources available on a single computer, virtualization and emulation have both been the target of a lot of research in the last years. Beyond their usual use case, they can be efficiently combined to build useful experimentation platforms, and are a promising tool to study peer-to-peer systems : they allow to use the real application on a large number of nodes in a configurable environment allowing reproduction of experiments.

This paper also contributes a simple network model different from the models usually used in network emulators to model the Internet : while those models concentrate on the core of the Internet and its routing interaction, P2PLab models the Internet from the end node's point of view.

This work also shows that, while most virtualization systems concentrate on providing a very accurate image of resources, a simpler approach might be sufficient in some cases. Our emulation platform only virtualizes what is needed to make the different virtual nodes look like real separate nodes from the outside : its network identity. This lightweight virtualization allows to maximize the virtualization ratio.

Our emulation platform, P2PLab, enabled us to perform some experiments on the BitTorrent peer-to-peer system. During those experiments, P2PLab proved to be scalable, easy to use and useful.

Figure 1 .

 1 Figure 1. Total calculation for several instances of the same process. Swap is not used.

Figure 2 . 6 Figure 3 .

 263 Figure 2. Total calculation for several instances of the same process. With more than 25 instances, swap is used.

Figure 4 .

 4 Figure 4. On each physical node, IP addresses for virtual nodes are configured as interface aliases.

Figure 6 .

 6 Figure 6. Example results of pings between nodes of the topology from figure 5.

Figure 7 .

 7 Figure 7. Overhead caused by Dummynet in a token ring

Figure 8 .

 8 Figure 8. Evolution of the download of the 160 clients.

  data received by clients (MB) time (s) 1 client per physical node 10 clients per physical node 20 clients per physical node 40 clients per physical node 80 clients per physical node

Figure 9 .

 9 Figure 9. P2PLab folding ratio : total amount of data when downloading of a 16 MB file with 160 clients.

Figure 10 .

 10 Figure 10. Evolution of the download of a 16 MB file between 5754 clients on a few selected clients.

Figure 11 .

 11 Figure 11. Number of clients having completed their download.

* This work has been done within the ID laboratory jointly supported by CNRS, INPG, INRIA, and UJF. Computer resources are provided by the Grid5000 platform (further information at http://www.grid5000.fr/).


Serena Janho Dit Hreich

Jonathan Benzaquen

Paul Hofman

Valérie Vouret- Craviari

Dr Valérie

To inhibit or to boost the ATP/P2RX7 pathway to fight cancer, that is the question

Keywords: purinergic signalling, P2X7, immunotherapies, tumors, antitumor immunity, ectonucleases

Despite new biological insights and recent therapeutic advances, many tumors remain at baseline during treatments. Therefore, there is an urgent need to find new therapeutic strategies to improve the care of patients with solid tumors. P2RX7 receptor (P2XR7), an ATPgated ion channel characterized by its ability to form large pore within the cell membrane, is described by most of the investigators as a "chef d'orchestre" of the antitumor immune response. The purpose of this review is to detail the recent information concerning different cellular mechanisms linking P2RX7 to hallmarks of cancer and to discuss different progresses in elucidating how activation of the ATP/P2RX7/NLRP3/IL-18 pathway is a very promising approach to fight cancer progression by increasing antitumor immune responses.

Introduction

According to the International Agency for Research on Cancer (IARC), the global burden of cancer reached 19.3 million new cases and 9.6 million deaths in 2020 (gco.iarc.fr). One in five men and one in six women worldwide will develop cancer in their lifetime, and one in eight men and one in 11 women will die from the disease (gco.iarc.fr). Nowadays, the five-year prevalence, corresponding to the total number of people living with cancer within five years of diagnosis, is estimated at 50.55 million.

At the beginning of cancer therapy, tumors were surgically removed, then came the time of chemotherapies -with molecules killing dividing cells -in association or not with radiation therapies. During the last 25 years, new approaches revolutionized clinical cancer care in daily practice. The first one relies on the understanding of oncogene dependent cell autonomous proliferation, leading to clinically relevant targeted therapies based on molecular targets. The second one relies on immune oncology and led to the development of immunotherapeutic strategies designed to turn on/off specific immune checkpoints to promote antitumor immunity. Other orientations for fighting cancer progression are continuously on progress and may open new doors for some therapeutic opportunities [START_REF] Hofman | New insights into the interaction of the immune system with nonsmall cell lung carcinomas[END_REF]. However, despite this new arsenal of therapeutics many patients still do not recover and/or relapse after a variable period of time, therefore testifying for the need of new drug development to fight cancer. In this review, we focus our attention on the P2X7 purinoreceptor (P2RX7), the only receptor being activated by high levels of extracellular adenosine triphosphate (eATP) and discuss recent progress in elucidating how activation of the ATP/P2RX7/NLRP3/IL-18 pathway can be a promising approach to fight cancer diseases by increasing antitumor immunity.

Exploring the tumor microenvironment to find new therapeutic strategies

It is now well documented that vascular networks and supporting cells are required to ensure the expansion of healthy tissues as well as the growth of tumors [START_REF] Farc | An overview of the tumor microenvironment, from cells to complex networks (Review)[END_REF]. Thus, in addition to directly target tumor cells, the non-cancerous cells present in the tumor microenvironment (TME) can also be viewed as relevant targets, even if this domain is now more and more complex [START_REF] Bagaev | Conserved pan-cancer microenvironment subtypes predict response to immunotherapy[END_REF]. These cells are mesenchymal supporting cells (epithelial, myoepithelial, fibroblasts, adipocytes), cells of the vascular system (endothelial, perivascular cells) and cells of the immune system (lymphoid and myeloid lineages). Altogether, they participate in the development of tumors as nicely illustrated by Egeblad and coauthors [START_REF] Egeblad | Tumors as organs: complex tissues that interface with the entire organism[END_REF]. Further, the continuous interplay between tumor cells and normal surrounding cells influences tumor growth. Indeed, it is well established that fibroblasts, the most abundant population within connective-tissue cells, fuel tumor growth [START_REF] Kuzet | Fibroblast activation in cancer: when seed fertilizes soil[END_REF]. Transforming growth factor beta (TGF-β), a growth hormone secreted by tumor and immune cells, favors the conversion of normal fibroblasts to carcinoma-associated fibroblasts by feeding an autocrine loop involving TGF-β and SDF-1 cytokines, which further lead to the production of tumor promoting chemokines [START_REF] Orimo | Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion[END_REF][START_REF] Kojima | Autocrine TGF-and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts[END_REF] and modulate the composition of immune cells within the TME where they can promote or inhibit tumor growth [START_REF] Gajewski | Innate and adaptive immune cells in the tumor microenvironment[END_REF][START_REF] Hanahan | Accessories to the Crime: Functions of Cells Recruited to the Tumor Microenvironment[END_REF][START_REF] Marvel | Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected[END_REF]. Is there a common intermediate between all these cells and if so, what could it be? An important step to answer this question was taken by Geoff Burnstock decades ago when he postulated that ATP is released by most cells and can be considered as an extracellular signaling molecule [START_REF] Burnstock | Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by nonadrenergic inhibitory nerves in the gut[END_REF]. This discovery was further sustained by the demonstration that eATP promotes calcium influx in macrophages [START_REF] Steinberg | Extracellular ATP4-promotes cation fluxes in the J774 mouse macrophage cell line[END_REF], via the seventh member of P2X purinoreceptor family, formerly identified as the cytolytic P2Z receptor [START_REF] Surprenant | The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7)[END_REF], which is described below. Finally, the importance of the ATP/P2RX7 pathway in the field of oncology was buried by the discovery that ATP is present at high levels (millimolar order) in tumor site [START_REF] Pellegatti | Increased level of extracellular ATP at tumor sites: In vivo imaging with plasma membrane luciferase[END_REF]. Of interest, all mammalian cells express P2RX7 mRNA and most of the cells express a functional receptor, i.e.

a receptor expressed at the cell membrane, with a channel and a macropore opening activities [START_REF] Benzaquen | Alternative splicing of P2RX7 pre-messenger RNA in health and diseases: Myth or reality?[END_REF]. In particular, functional P2RX7 was described in cells that composed the TME, such as fibroblasts [START_REF] Solini | Human primary fibroblasts in vitro express a purinergic P2X 7 receptor coupled to ion fluxes , microvesicle formation and IL-6 release[END_REF], endothelial cells [START_REF] Oliveira Sd Dos | Endothelial P2X7 receptors' expression is reduced by schistosomiasis[END_REF], dendritic cells [START_REF] Georgiou | Human Epidermal and Monocyte-Derived Langerhans Cells Express Functional P2X 7 Receptors[END_REF][START_REF] Baroni | Stimulation of P2 ( P2X 7 ) receptors in[END_REF][START_REF] Idzko | Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors[END_REF], monocytes/macrophages [START_REF] Gu | Expression of P2X7 purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X7 receptors[END_REF][START_REF] Chiozzi | Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X 7 Receptor[END_REF], lymphocytes [START_REF] Borges | The purinergic receptor P2RX7 directs fitness of long-lived memory CD8 + T cells[END_REF], M-and G-MDSC [START_REF] Bianchi | ATP/P2X7 axis modulates myeloidderived suppressor cell functions in neuroblastoma microenvironment[END_REF] and neutrophils [START_REF] Christenson | Serum amyloid A inhibits apoptosis of human neutrophils via a P2X7-sensitive pathway independent of formyl peptide Abstract : Neutrophil apoptosis is important for the termination of inflammatory reactions , in that[END_REF] as illustrated in Figure 1. In addition, expression of a functional P2RX7 was found in both solid [START_REF] Deli | Functional genomics of calcium channels in human melanoma cells[END_REF][START_REF] Solini | Increased P2X 7 Receptor Expression and Function in Thyroid Papillary Cancer : A New Potential Marker of the Disease ?[END_REF][START_REF] Fg | Paracrine Stimulation of P2X7 Receptor by ATP Activates a[END_REF][START_REF] Santos | Potential role of P2X7R in esophageal squamous cell carcinoma proliferation[END_REF] and liquid cancers [START_REF] Adinolfi | Brief report P2X 7 receptor expression in evolutive and indolent forms of chronic B lymphocytic leukemia[END_REF][START_REF] Zhang | Expression of P2X7 in human hematopoietic cell lines and leukemia patients[END_REF][START_REF] Chong | Abnormal expression of P2X family receptors in Chinese pediatric acute leukemias[END_REF].

The P2X7 purinoreceptor: A signaling hub underpinning tumor onset

The P2X7 purinoreceptor (also called P2X7R or P2RX7) belongs to the purinergic receptor family, which is divided into two major families: P2X and P2Y [START_REF] Jacob | Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses[END_REF]. These receptors have attracted growing interest since the discovery that inflammatory and cancer tissues contain high levels of eATP and adenosine, its immunosuppressive degradation product [START_REF] Pellegatti | Increased level of extracellular ATP at tumor sites: In vivo imaging with plasma membrane luciferase[END_REF][START_REF] Allard | Immunosuppressive activities of adenosine in cancer[END_REF].

Depending on the receptor involved, extracellular purines orchestrate either immune stimulation or immune suppression of host cells, as well as proliferation or cytotoxicity of tumor cells [START_REF] White | P2 receptors and cancer[END_REF].

P2RX7 is an ATP-gated ion channel composed of three protein subunits (encoded by the P2RX7 gene). Each monomer is composed of two transmembrane domains that are linked by a large extracellular domain made of 285 amino acids; The N and C termini are intracellular, with a very long C-terminus, comprising various moieties such as trafficking and lipid interaction domains and death domain, as nicely illustrated in [START_REF] Kopp | P2X7 Interactions and Signaling -Making Head or Tail of It[END_REF]. This particular feature makes P2RX7 unique, as well as the fact that it is activated by high concentrations of eATP, implicating that it remains active where and when the other P2X receptors showed no current upon agonist application [START_REF] North | Pharmacology of Cloned P2X Receptors[END_REF]. Activated P2RX7 stimulates the entry of Ca 2+ and Na + but also the efflux of K + . Intracellular Ca 2+ is an ubiquitous second messenger known to control various cellular functions such as cell homeostasis, gene expression, cell survival/death, exo and endocytosis and contraction [START_REF] Bagur | Intracellular Ca 2+ Sensing: Its Role in Calcium Homeostasis and Signaling[END_REF]. The ATP/P2RX7-induced increase of intracellular Ca 2+ levels are highly relevant in multiple cell responses. In macrophages, P2RX7-dependant calcium entry and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway have been shown to produce reactive oxygen species (ROS) [START_REF] Martel-Gallegos | Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2[END_REF], but also lipase activation and blebbing [START_REF] Kopp | P2X7 Interactions and Signaling -Making Head or Tail of It[END_REF]. In the nervous system, P2RX7 activation has been demonstrated to modulate sodium currents and intrinsic neuron excitability [START_REF] Del Puerto | ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury[END_REF]. Ion channel function is independent of the long C-terminus, as its deletion or truncation do not alter it [START_REF] Smart | P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region[END_REF]. Accordingly, the truncated P2RX7B isoform retains some ATPinduced channel activity and expression of P2RX7B in HEK293 cells increases stimulates growth [START_REF] Adinolfi | Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor[END_REF]. However, it is has been shown that domains lying outside of the pore also participate in the regulation of Ca 2+ flux [START_REF] Liang | Quantifying Ca2+ current and permeability in ATP-gated P2X7 receptors[END_REF].

In addition to increase intracellular Ca 2+ concentrations, P2RX7 activation leads to a decrease in intracellular K + which has been recognized as one of the main activators of NLRP3 inflammasome [START_REF] Pétrilli | Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration[END_REF]. Activation of NLRP3 inflammasome is intimately linked to innate immunity being involved in the production of two key potent proinflammatory cytokines, IL-1β and IL-18 which have also been described to be important in tumor onset as they stimulate the production of Interferon gamma (IFN-) by immune effector cells (T lymphocytes, Natural Killer) [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1Β-dependent adaptive immunity against tumors[END_REF][START_REF] Ma | ATP-dependent recruitment, survival and differentiation of dendritic cell precursors in the tumor bed after anticancer chemotherapy[END_REF]. In addition, activation of NLRP3 inflammasome leads to the formation of pores in the plasma membrane, via Gasdermin D [START_REF] Pelegrin | P2X7 receptor and the NLRP3 inflammasome: Partners in crime[END_REF], that results ultimately to pyroptotic cell death.

Yet, P2RX7 activation by itself is also associated with the formation of a large pore. After decades of research and various hypothesis (mainly pore dilatation and accessory proteins), it is now well admitted that the pore formation is intrinsic of the channel activity [START_REF] Harkat | On the permeation of large organic cations through the pore of ATP-gated P2X receptors[END_REF][START_REF] Pippel | Localization of the gate and selectivity filter of the full-length P2X7 receptor[END_REF] and can be regulated by the C-terminus which interacts with phosphatidylglycerols and sphingomyelins of the cell membrane to facilitate cell permeability [START_REF] Karasawa | The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition[END_REF]. Karasawa and co authors also elucidated why the truncated P2RX7 was devoided of pore formation by demonstrating that the C-terminal Cys-rich region, which is deleted in the truncated isoform, counteracts the inhibitory effect of cholesterol, as previously discussed [START_REF] Benzaquen | Alternative splicing of P2RX7 pre-messenger RNA in health and diseases: Myth or reality?[END_REF][START_REF] Virgilio | The Elusive P2X7 Macropore[END_REF].

As already mentioned, the long intracellular C-terminus of P2RX7 is required for most of the signaling pathways induced by the ATP/P2RX7 axis. We will focus here on P2RX7-induced cell responses that are related to hallmarks of cancer (Figure 2) as described by Hanahan et al.

P2RX7 and sustained cell proliferation

Sustained proliferation is a characteristic of tumor cells and is under the control of growth factors that bind receptors with intracellular tyrosine kinase domains which further stimulate intracellular signaling pathways leading to cell proliferation. ATP, the natural ligand of P2RX7 does not belong to the growth factor family and no tyrosine kinase domains have been described in the C-terminus of P2RX7. However, several reports support the notion that P2RX7 activation by low concentration of ATP, as compared to concentrations inducing cell death, leads to cell proliferation [START_REF] Adinolfi | Expression of P2X7 receptor increases in vivo tumor growth[END_REF][START_REF] Haanes | The P2X7 Receptor Supports Both Life and Death in Fibrogenic Pancreatic Stellate Cells[END_REF][START_REF] Giannuzzo | The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells[END_REF][START_REF] Zhang | Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/βcatenin and mTOR/HIF1α/VEGF signaling[END_REF][START_REF] Zhang | PI3K/Akt/GSK-3β signal pathway is involved in P2X7 receptor-induced proliferation and EMT of colorectal cancer cells[END_REF]. Even in the absence of tyrosine kinase domains, stimulation of the ATP/P2RX7 axis can trigger kinase activation. For instance, Cellular Sarcoma Tyrosine Kinase (c-Src), Mitogen Activated Protein Kinases (ERK1/2, p38, JNK), Phosphoinositide 3-Kinase (PI3Ks)/Protein Kinase B (PKB/Akt) and Protein Kinases C (PKC) have been shown to be activated by P2RX7 [START_REF] Kopp | P2X7 Interactions and Signaling -Making Head or Tail of It[END_REF]. However, no proof of direct interactions exists, and it is likely that kinase activation is mediated via Ca 2+ and/or colocalization of P2RX7 and these kinases in lipid rafts. Accordingly, a lipid interaction motif has been described in the C-terminus of P2RX7 (amino acid 574-589) and P2RX7 move toward lipid rafts in response to ATP [START_REF] Denlinger | Cutting Edge: The Nucleotide Receptor P2X 7 Contains Multiple Protein-and Lipid-Interaction Motifs Including a Potential Binding Site for Bacterial Lipopolysaccharide[END_REF][START_REF] Gonnord | Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts[END_REF][START_REF] Robinson | Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization[END_REF][START_REF] Murrell-Lagnado | Regulation of P2X Purinergic Receptor Signaling by Cholesterol[END_REF]. In addition, the proteomic characterization of P2RX7 protein complex in HEK293 cells expressing the rat P2RX7 isoform identified 11 proteins, some of them being involved in cytoskeletal rearrangement [START_REF] Kim | Proteomic and functional evidence for a P2X7 receptor signalling complex[END_REF], a cell response directly linked to cell proliferation. However, the effect of P2RX7 on sustained cell proliferation is controversial. For instance, in glioma tissue, inhibition of P2RX7 by ShRNA increased the expression of EGF and phosphor-EGF protein [START_REF] Fang | P2X7R suppression promotes glioma growth through epidermal growth factor receptor signal pathway[END_REF] and it was reported that reduced expression of P2RX7 may be related to the development of several cancers [START_REF] Benzaquen | Alternative splicing of P2RX7 pre-messenger RNA in health and diseases: Myth or reality?[END_REF][START_REF] Feng | Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate[END_REF][START_REF] Feng | A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization[END_REF][START_REF] Lara | P2X7 in Cancer: From Molecular Mechanisms to Therapeutics[END_REF].

P2RX7 and growth suppressor evasion

To hijack programs that negatively regulate cell proliferation is as important as promoting cell proliferation. To our knowledge P2RX7 does not inhibit tumor suppressors such as TP53 or retinoblastoma-associated (RB) proteins. On the contrary, it was reported that the ATP/P2RX7 axis supports apoptosis and necrosis by increasing p53 protein levels in mesangial [START_REF] Schulze-Lohoff | Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X 7 receptors[END_REF] and human cervical cancer cells [START_REF] Mello P De | Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells[END_REF].Whether the death domain localized in the C-terminus of P2RX7 [START_REF] Denlinger | Cutting Edge: The Nucleotide Receptor P2X 7 Contains Multiple Protein-and Lipid-Interaction Motifs Including a Potential Binding Site for Bacterial Lipopolysaccharide[END_REF] is involved in this effect has not been studied. Yet, activation of P2RX7 is associated with mechanisms and or cellular responses leading to growth suppressor evasion since tumors grew faster in p2rx7 -/-mice [START_REF] Hofman | Genetic and pharmacological inactivation of the purinergic P2RX7 receptor dampens inflammation but increases tumor incidence in a mouse model of colitis-associated cancer[END_REF][START_REF] Adinolfi | Accelerated tumor progression in mice lacking the ATP receptor P2X7[END_REF], an effect depending on host immune cells, as discussed below.

P2RX7 and cell death resistance

Resisting to apoptosis, a programmed cell death viewed as a natural barrier to cancer, is one of the characteristics of tumor cells. Depending on the cellular origin, the ATP/P2RX7 axis induces or inhibits apoptosis. In colorectal cancer cells a pharmacological inhibitor of P2RX7 induced apoptosis [START_REF] Zhang | PI3K/Akt/GSK-3β signal pathway is involved in P2X7 receptor-induced proliferation and EMT of colorectal cancer cells[END_REF]. In the MCF7 breast cancer model, decrease of P2RX7 expression with ShRNA induced cell apoptosis [START_REF] Tan | Expression of P2X7R in breast cancer tissue and the induction of apoptosis by the gene-specific shRNA in MCF-7 cells[END_REF]. By contrast, in acute myeloid leukemia, the ATP/P2RX7 axis induced apoptosis [START_REF] Salvestrini | Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells[END_REF]. Whether some evidence highlight a relationship between P2RX7induced signaling and apoptosis, it does not seem that these interactions act as a barrier to cancer. The same conclusion can be drawn for autophagy -an ancestral pathway which maintains cellular homeostasis by degrading long-lived proteins and removing unwanted or unnecessary intracellular components -which represents another cellular response that needs to be circumvented for tumor development. P2RX7-induced signaling pathway has been shown to negatively regulate the autophagic flux through the impairment of lysosomal functions in microglial cells [START_REF] Takenouchi | The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells[END_REF]. This pathway, in association with HSP90, was also linked to autophagic cell death in myoblasts and was triggered by large pore formation [START_REF] Young | A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90[END_REF]. In contrast, P2RX7-induced necrosis may be of particular relevance since it allows the release of growth factors which foster proliferation and invasiveness of cancer cells [START_REF] Giannuzzo | The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells[END_REF], but also immunomodulatory cytokines as discussed below. Finally, it has been shown that tumor cells express a non-conformal receptor (nfP2RX7) which is unable to induce cell death but retains calcium channel activity and therefore could promote tumor growth [START_REF] Barden | Non-Functional P2X7: A Novel and Ubiquitous Target in Human Cancer[END_REF][START_REF] Gilbert | ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival[END_REF]. The exact nature of this nfP2RX7 still remains an enigma but it may be linked to P2RX7B expression, as discussed in the P2RX7 and genomic instability chapter.

P2RX7 and replicative immortality

The unlimited replicative property of cancer cells is required to generate macroscopic tumor. P2RX7 could favor cell proliferation (see above), but whether P2RX7 could be linked to replicative immortality is currently unknown.

P2RX7 and angiogenesis

The formation of new vessels from pre-existing ones, called neo angiogenesis, is required to fuel tumors with oxygen and nutriments. The architecture of the tumor vasculature is abnormal, i.e. leaky, tortuous, fragile, and blind-ended with few or no perivascular cells. Such defectiveness leads to hypoxia and acidity in the TME, further reinvigorating tumor aggression. Therefore, normalization of abnormal tumor vasculature, resulting in enhanced drug delivery, is as important as inhibiting neovascularization. Angiogenesis is under the control of a growth factor called Vascular Endothelial Growth Factor (VEGF) which binds to the VEGF receptor (VEGFR2) to increase endothelial cell proliferation. P2RX7 has been reported to promote VEGF release from human monocytes and rat glioma cells [START_REF] Hill | Extracellular ATP May Contribute to Tissue Repair by Rapidly Stimulating Purinergic Receptor X7-Dependent Vascular Endothelial Growth Factor Release from Primary Human Monocytes[END_REF][START_REF] Wei | Expression and function of the P2X7 receptor in rat C6 glioma cells[END_REF].

Tumors obtained after engraftment of human embryonic kidney cells expressing P2RX7 are more angiogenic than tumors obtained with control cells [START_REF] Adinolfi | Expression of P2X7 receptor increases in vivo tumor growth[END_REF]. Mechanistically, it was proposed that the ATP/P2RX7 axis activates the PI3K/AKT and NFkB pathways which further lead to increased production of VEGF and increased-vessel density [START_REF] Adinolfi | Expression of P2X7 receptor increases in vivo tumor growth[END_REF][START_REF] Amoroso | The P2X7 receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network: evidence in experimental neuroblastoma[END_REF][START_REF] Yang | P2X7R promotes angiogenesis and tumour-associated macrophage recruitment by regulating the NF-κB signalling pathway in colorectal cancer cells[END_REF]. Therefore, in tumor mice models, P2RX7 activation likely stimulates angiogenesis. However, to our knowledge no study in human cancer demonstrates a link between P2RX7 expression and neo angiogenesis.

P2RX7 and invasion and metastasis

Malignant cells possess the ability to invade surrounding tissues and metastasize. Importantly, the metastatic spread of the primary tumor accounts for over 90% of patient mortality associated with solid cancers [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Metastasis is closely associated to the epithelialmesenchymal transition (EMT), a cellular response being under the control of transcription factors belonging to the Twist and Snail families, which in turn inhibit the expression of epithelial marker (E-cadherin) and increase the expression of mesenchymal markers (vimentin, N-cadherin) [START_REF] Lambert | Linking EMT programmes to normal and neoplastic epithelial stem cells[END_REF]. P2RX7 is highly expressed and fully functional in the highly invasive human breast cancer cells MDA-MB-435s; its activation leads to the acquisition of a pro migratory phenotype and enhanced cancer cell invasiveness in vitro and in vivo [START_REF] Jelassi | P2X7 receptor activation enhances SK3 channels-and cystein cathepsin-dependent cancer cells invasiveness[END_REF][START_REF] Jelassi | Anthraquinone emodin inhibits human cancer cell invasiveness by antagonizing P2X7 receptors[END_REF] thanks to the release of extracellular matrix degrading proteases. In lung cancer cells, the ATP/P2RX7 axis induced an autocrine regulation of TGF-β-induced cell migration [START_REF] Takai | Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells[END_REF]. Further, P2RX7 effect on cell migration and invasion was observed in response to ATP in pancreatic ductal adenocarcinoma cells [START_REF] Giannuzzo | The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells[END_REF], but also in the human T47D breast cancer cells and colon cancer cells, where activation of the PI3K/AKT/GSK3 pathway in response to ATP regulates Ecadherin expression and EMT [START_REF] Zhang | PI3K/Akt/GSK-3β signal pathway is involved in P2X7 receptor-induced proliferation and EMT of colorectal cancer cells[END_REF][START_REF] Xia | P2X7 receptor stimulates breast cancer cell invasion and migration via the AKT pathway[END_REF]. Whereas many reports highlight the ability of P2RX7 to induce cell invasion and migration in cultured tumor cells, only one study demonstrates its implication in metastasis, considering that metastases correspond to secondary tumors growing at distance of the primary tumor. Using the mammary 4T1 cancer mouse model, it was shown that P2RX7 expression promotes tumor growth and metastasis development, both of them being reduced when mice were treated with specific P2RX7 antagonists [START_REF] Brisson | P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment[END_REF].

Mechanistically, P2X7R expression enhances the ECM-degradative activity of invadopodia and triggers the acquisition of a more aggressive phenotype, characterized by activation of Cdc42Rho-GTPase, remodeling of F-actin, elongation of cells and formation of filopodia.

Further, a higher expression of P2RX7 has been documented in bronchoalveolar lavage cells of tumors with distant metastasis [START_REF] Schmid | Altered purinergic signaling in the tumor associated immunologic microenvironment in metastasized non-small-cell lung cancer[END_REF] and in B16 melanoma-induced lung metastasis in mice.

However and in contradiction with the above findings, more lung tumors were observed with B16 melanoma cells injected in p2rx7 -/-mice, suggesting that endogenous P2RX7 activity held by host cells restrains B16 migration and invasion [START_REF] Adinolfi | Accelerated tumor progression in mice lacking the ATP receptor P2X7[END_REF]. Undoubtedly, additional experiments are required to fully appreciate the role of P2RX7 in invasion and metastasis depending on the cancer origin.

P2RX7 and genomic instability

Genomic instability of cancer cells favors the selection of subclones demonstrating growth advantage and nowadays study of tumor mutational load is proposed as a sensitive and quantitative clinical marker that can help predict responses to cancer treatment. To our knowledge, P2RX7 was not reported to directly induce DNA mutations. However, several number of missense mutations are present in human P2RX7 and until now 16 Single Nucleotide Polymorphisms (SNPs) have been characterized in the coding region of human P2RX7 gene [START_REF] Sluyter | Significance of p2x7 receptor variants to human health and disease[END_REF]. These mutations affect P2RX7 activity. For instance, the polymorphism in exon 13 at the +1513 position (Glu496Ala substitution, corresponding to SNP rs3751143) has been shown to eradicate the function of this receptor and to be associated with papillary thyroid cancer, hepatocarcinoma and chronic lymphocytic leukemia [START_REF] Cabrini | A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X 7 Receptor of Human Leukemic Lymphocytes[END_REF][START_REF] Dardano | 1513A>C Polymorphism in the P2X7 Receptor Gene in Patients with Papillary Thyroid Cancer: Correlation with Histological Variants and Clinical Parameters[END_REF][START_REF] Duan | Association Between P2RX7 Gene and Hepatocellular Carcinoma Susceptibility: A Case-Control Study in a Chinese Han Population[END_REF][START_REF] Virgilio | The P2X7 receptor of CLL lymphocytes-a molecule with a split personality[END_REF]. However, a recent meta-analysis studying the link between rs3751143 and cancer risk minored these results [START_REF] Wang | Predicted the P2RX7 rs3751143 polymorphism is associated with cancer risk: a meta-analysis and systematic review[END_REF]. Nevertheless, in the field of hematological malignancies, P2RX7 and its genetic variants are now considered as biomarkers and potential therapeutic tools [START_REF] Marchi | P2X7 Receptor in Hematological Malignancies[END_REF].

In non-small cell lung cancer P2RX7 expression and the SNP rs3751143 does not impact tumor onset [START_REF] Boldrini | P2X7 protein expression and polymorphism in non-small cell lung cancer (NSCLC)[END_REF], whereas this SNP was associated with reduced metastatic free survival in anthracyclines treated breast cancer [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1Β-dependent adaptive immunity against tumors[END_REF]. On the contrary, a genetic statistical interaction between P2RX7 (rs3751143, rs208294) and VEGFR-2 (rs2071559, rs11133360) genotypes may identify a population of prostate cancer patients with a better prognosis [START_REF] Solini | Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients[END_REF]. In addition to SNPs, the pre-messenger RNA of P2RX7 is subjected to alternative splicing leading to the formation of 12 P2RX7 mRNA variants [START_REF] Benzaquen | Alternative splicing of P2RX7 pre-messenger RNA in health and diseases: Myth or reality?[END_REF][START_REF] Cheewatrakoolpong | Identification and characterization of splice variants of the human P2X7 ATP channel[END_REF][START_REF] Skarratt | A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function[END_REF]. The function of only four of them, P2RX7B with a deletion in the C-terminal domain (ΔCt) leading to a loss of large pore forming function, P2RX7H with a deletion in the first transmembrane domain (ΔTM1) leading to a loss of ion channel function, P2RX7J with a deletion in the second transmembrane domain leading to a negative dominant isoform and the recently described P2RX7L, characterized by skipping of exons 7 and 8 leading to deletion of amino acids in the ATP binding site, have been studied in more details [START_REF] Adinolfi | Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor[END_REF][START_REF] Feng | A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization[END_REF][START_REF] Skarratt | A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function[END_REF][START_REF] Giuliani | Trophic activity of human P2X7 receptor isoforms A and B in osteosarcoma[END_REF][100]. The truncated P2RX7J was first described to antagonize P2RX7A function in cancer cervical cells, this effect was associated with apoptosis-inhibitory actions [START_REF] Feng | A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization[END_REF]. More recently, the P2RX7B splice variant has been shown to be differentially upregulated in immune cells of human Non-Small Cell Lung Cancer. This overexpression correlates with a default in the activity of P2RX7 in immune cells and lung tumors expressing P2RX7B are less inflamed. Therefore, considering that expression of P2RX7B in lung tumors correlated with both an alteration in the P2RX7 function and the lesser infiltrated tumor phenotype (also called "cold" tumor), it was proposed that P2RX7B participates in lung tumor development [101]. A similar conclusion was drawn in patients with AML. In those patients expression levels of both P2RX7A and B mRNA are increased and daunorubicin, the main chemotherapeutic for AML, only reduced the expression levels of P2RX7A, whereas P2RX7B levels remained high, which correlate with cells more prone to survive [102]. One may hypothesize that the heterotrimers composed of P2RX7A and P2RX7B isoforms may correspond to the nfP2RX7, since they shared the same biological activities e.g., no large pore formation and low Ca2+ channel activity [START_REF] Gilbert | ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival[END_REF].

P2RX7 and reprogramming energy metabolism

P2RX7 and immune responses related to tumor onset

Beside efficiently fighting infections, the immune system is also programmed to eliminate cancer cells that ultimately evade immune surveillance via checkpoint inhibitor expression and secretion of immunosuppressive factors but also survival and migration factors. [107, 108]. As discussed above, P2RX7's functions have been extensively studied in immune cells. We will focus here on P2RX7's immune populations known to be involved in tumor onset where p2rx7 -/-mice developed more tumors and tumors were more infiltrated with Tregs than tumors grown in WT mice [START_REF] Hofman | Genetic and pharmacological inactivation of the purinergic P2RX7 receptor dampens inflammation but increases tumor incidence in a mouse model of colitis-associated cancer[END_REF]127]. Such results could be explained by the fact that P2RX7 controls Treg homeostasis and in absence of P2RX7, Treg death is decreased [128,129].

Finally, whereas the level of P2RX7 expression in human MDSCs has not yet been reported, it was shown in the neuroblastoma mice model that the ATP/P2RX7 axis increases immunosuppressive functions of MDSCs [130]. In conclusion, while it is indisputable that P2RX7 modulates immune cell functions, the contradicting results found in the literature make it is difficult to predict whether P2RX7, by itself, impinges or enhances antitumor immunes responses.

Should we target P2RX7 in cancer biology?

This is indeed THE QUESTION, and as evidenced in previous chapters, this is a puzzling question (Figure 3). Should we inhibit P2RX7 in order to blunt its proliferative effect on cancer cells? However, this scenario may be dangerous since it may also inhibit antitumor immune responses sustained by P2RX7? Should we activate P2RX7 in order to boost its cytotoxic effect on tumor cells? But is P2RX7 really cytotoxic on tumor cells, since we know that many tumors express a nfP2RX7? If we activate P2RX7, would it really enhance antitumor immune responses or would it boost the function of immunosuppressive cells that are recruited in the TME?

Since the notion of immunosurveillance emerges only recently in the field of cancer biology

[131] and because big pharma were more prone to develop inhibitors, especially P2RX7 antagonists that may treat inflammatory disorders, numerous small pharmacological inhibitors and biologics were tested in preclinical and clinical studies as reviewed in [START_REF] Lara | P2X7 in Cancer: From Molecular Mechanisms to Therapeutics[END_REF]. In brief, most of the preclinical studies were based on transplantation in immunodeficient or syngeneic immunocompetent mice of various tumor cell lines (e.g., HEK293, neuroblastoma, colon carcinoma, melanoma, glioma, osteosarcoma and myeloid leukemia) endowed with endogenous or exogenous expression of P2RX7, where tumor growth was significantly reduced by treatment with AZ10606120, A438079 or A740003, three well described antagonists of P2RX7 [START_REF] Adinolfi | Expression of P2X7 receptor increases in vivo tumor growth[END_REF][START_REF] Giannuzzo | The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells[END_REF][START_REF] Zhang | Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/βcatenin and mTOR/HIF1α/VEGF signaling[END_REF][START_REF] Adinolfi | Accelerated tumor progression in mice lacking the ATP receptor P2X7[END_REF]127,132]. However, an acceleration of tumor growth was also described when tumor cell lines were transplanted in p2rx7 KO mice or with chemically -induced in situ colon tumors [START_REF] Hofman | Genetic and pharmacological inactivation of the purinergic P2RX7 receptor dampens inflammation but increases tumor incidence in a mouse model of colitis-associated cancer[END_REF][START_REF] Adinolfi | Accelerated tumor progression in mice lacking the ATP receptor P2X7[END_REF]. These results highlight the importance of P2RX7 expressing host cells in tumor onset and suggest that the levels of expression and functionality of P2RX7 in tumor cells is a critical issue that have to be considered before the use of antagonists to treat tumor. As far as we know, no clear benefits to patients have been reported so far and this lack of effect may be related to the fact that inflammatory tissues contain high levels of ATP, a condition that does not favor the binding of antagonist to the allosteric binding site [133]. The use of biologics may be an alternative. In particular, application of an antibody that specifically recognizes the E200 epitope of nfP2RX7 appears to be promising for the local treatment of the primary basal cell carcinoma lesion [134].

We hypothesized that increasing P2RX7's dependent antitumor immune response by enhancing its activation would inhibit tumor growth. We developed positive allosteric modulators (PAMs) of P2RX7, i.e. molecules that enhances P2RX7 activity in the presence of ATP. HEI3090 was selected as the most potent PAM that enhances P2RX7's activities only in the presence of ATP and showed that it efficiently inhibits tumor growth.

Perspectives

Undoubtably, targeting P2RX7 to treat cancer is a promising strategy and recent data from several laboratories highlighted the capacity of the ATP/P2RX7/NLRP3/IL-18 axis to activate antitumor immune responses. However, pivotal questions remain to be answered. Among them, how do the new pharmacological positive modulator of P2RX7 (HEI3090) enhances the receptor activity? Does it bind to the receptor itself and if so, where? Is HEI3090 active on the nfP2RX7? And if so, which and how, P2RX7-dependent biological responses are going to be regulated? Would it be interesting to combine HEI3090 with the new anti CD39 antibody in order to maximize the activation of the ATP/P2RX7/NLRP3/IL-18 axis and the consecutive antitumor immune response? Would the other non-nucleotide agonists of P2RX7 (i.e. ginsenoside, LL37 [138, 139] will also boost the antitumor immune responses supported by the ATP/P2RX7 axis? and if so, could we combine them with HEI3090 and immunotherapies?

What would be the effect of an agonist of P2RX7 on inflammatory pain and IL-1β-induced cachexia, knowing that P2RX7 antagonists not only efficiently inhibit the production of IL-1β but also reduced cancer-induced bone pain [140, 141]? In this perspective, the use of HEI3090 may be of particular interest, since we showed in transplantable and oncogene-driven tumor models that it increases seric IL-18 levels with no effect on IL-1β. We believe that these points will be resolved in a near future, and in addition, we are strongly convinced that their resolution will bring new innovative strategies to fight cancers.

Otto

  Warburg was the first to described that cancer cells can reprogram their glucose metabolism, thanks to glycolysis, even in the presence of oxygen [103]. In the heterotopic HEK293 cell system, expression of P2RX7 leads to the regulation of numerous glycolytic markers conferring to this receptor the power to control cellular energy metabolism [104]. In a human cohort which includes more refined measures of glucose homeostasis, P2RX7 has been linked to glucose regulation [105]. Accordingly, it was shown that P2RX7 loss of function in mice produces defective energy homeostasis [106]. To our knowledge, no study demonstrated that P2RX7 directly regulates energy metabolism of cancer cells. Yet, numerous important studies link P2RX7 to immunometabolism as discussed below.

  [109]. An efficient anticancer immune response is normally sufficient to kill cancer cells. On one hand, soluble neoantigens express by cancer cells are recognized by circulating Antigen Presenting Cells (APC) such as dendritic cells (DCs) and macrophages. Then, APC present the tumor antigens on MHC-I and MHC-II molecules to T cells (CD8+, CD4+) leading to priming and activation of effector T cell responses which traffic to tumor bed, recognize tumor antigen and kill tumor cells. On the other hand, the Natural Killer (NK) cells can directly kill tumor cells by using cytolytic granules and death receptors and by potentiating adaptive immunity through the production of cytokines such as Interferon gamma (IFN-) [110]. These antitumor immune responses are blunted by immunosuppressive cells such as regulatory T cells (Treg), and myeloid derived suppressor cells (MDSC) [111]. Both macrophages and DCs from human origin express P2RX7 which is able to modulate cytokine production in response to eATP and LPS [112-115]. The potential of the ATP/P2RX7 axis to enhance antitumor immune responses was suggested by Kroemer's team after the demonstration that eATP attracts DC precursors into the tumor bed, facilitates their permanence in the proximity of dying cells and promotes their differentiation into mature DCs endowed with the capacity of presenting tumor-associated antigens [116]. In addition, P2RX7 activation in macrophages and DCs induces NLRP3 leading to increased production of IL-1β and IL-18. Both pro-tumorigenic and anti-tumorigenic roles of NLRP3 inflammasome have been described in cancers [117]. This double-edged sword effect may depend on the quantity of cytokine produced. For instance, more IL-18 will trigger a more efficient antitumor immune response [118]. Recent studies involving patient with acute myeloid leukemia revealed that P2RX7 mRNAs (A and B) are upregulated in immune cancer versus immune non cancer cells and AML patients with higher levels of P2RX7 mRNA show a lower survival rate [102, 119]. Further, engraftment of human myeloid cell lines in immunodeficient mice leads to tumor growth and treatment with specific antagonists of P2RX7 decreases the size of the tumors and increases mice survival rate. The importance of the ATP/P2RX7 axis in leukemic models was further improved by the discovery that P2RX7 is highly expressed in leukemia initiating cells where it promotes AML development by sustaining Ca2+-dependent cell signaling [120]. Human lymphocytes also express P2RX7 and demonstrated a growth promoting activity [121, 122]. Of note, at least in mice, P2RX7 expression levels is heterogenous across immune cells. Indeed, it is highly expressed by macrophages, DCs and monocytes while NK cells, CD8+ and CD4+foxp3-T cells, neutrophils and eosinophils express very low levels [123]. Yet, it was shown that P2RX7 is required for the establishment, maintenance and functionality of longlived memory CD8+ T cells (TRM), an effect that depends on P2RX7-dependent metabolic fitness [124]. Interestingly, in a melanoma mice model, TRM can actively suppress cancer progression [125]. P2RX7 is also expressed by human Tregs. It was shown that esophageal carcinoma from patients expressing the loss-of-function allele of P2RX7 are more infiltrated with Tregs than tumors from patients bearing the WT allele of P2RX7 [126]. The same tendency was observed in the colitis associated cancer mice model and melanoma models,

  HEI3090 enhances antitumor immune responses by boosting the ATP/P2RX7/NLRP3/IL-18 pathway in DCs, sensitizes tumor to anti PD-1 immunotherapy and triggers long lasting antitumor immune responses [135]. Our bet looks like a successful story, which is not so surprising considering that anti CD39 antibody was recently shown to promote the eATP/P2RX7/NLRP3/IL18-driven antitumor responses [123]. In addition, activation of this pathway with anti CD39 monoclonal antibody in myeloid cells activates effector function of NK cells and suppresses experimental and spontaneous metastases in a tumor of different origins [136], therefore bringing new hopes for the treatment of cancers. However, more work is needed to understand which immune cells should be targeted to kill tumor cells in order to reconciliate data showing a benefit to activate the ATP/P2RX7 axis in specific immune cells (macrophages, DCs, NK) and data showing that the lack of P2RX7 in tumor-specific T cells reduced tumor growth [137]. Further, we need to understand why tumor fate is different in p2rx7 null mice and mice treated with an antagonist of P2RX7 [127].

Figure 1 :

 1 Figure 1 : P2RX7 expression in eukaryotic cells

Figure 2 :

 2 Figure 2 : P2RX7-induced cell responses related to hallmarks of cancer

Figure 3 :

 3 Figure 3 : The yin and yang of P2RX7's modulators

  Figure 3

Funding VV-C was supported by grants from Institut National du Cancer (INCa # 2015-137), Canceropole PACA, Bristol-Myers Squibb Foundation for Research in Immuno-Oncology, the French Government (National Research Agency, ANR through the "Investments for the Future": program reference #ANR-11-LABX-0028-01) and the Centre National de la Recherche Scientifique (CNRS). SJH was supported by the "Ligue Nationale Contre le Cancer" and the "Fondation pour la recherche médicale # FDT202106013099" JB was supported by the "Plan Cancer: Formation à la recherche Translationnelle en cancérologie ».

Conflicts of interest:

All authors declare they have no conflicts of interest.

Ethical approval: This review does not contain any studies with participants or animals performed by any of the authors.