P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: role of the A2A/CD39/CD73 axis

P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: role of the A2A/CD39/CD73 axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: role of the A2A/CD39/CD73 axis Elena Adinolfi, Anna Pegoraro, Elena De Marchi, Luigia Ruo, Michele Zanoni, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5287461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Aug, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Extracellular vesicle-driven cancer metastasis represents a therapeutic challenge due to the lack of effective blocking drugs. This study reveals a unique mechanism involving the P2X7 receptor and the A2A/CD39/CD73 axis, which affects ATP and adenosine levels in cancer via vesicular release, thereby enhancing metastasis. It also introduces a novel P2X7-based therapeutic approach to target tumor vesicular release. Indeed, activation of P2X7 on colon carcinoma cells induced the release of extracellular vesicles carrying P2X7, A2A, CD39, and CD73, resulting in significantly elevated ATP and adenosine levels within the tumor microenvironment. These vesicles enhanced colon carcinoma metastatic potential and systemic IL-17 production when administered in vivo , effects that were successfully mitigated through P2X7 antagonism, which also reduced A2A levels in the metastatic niche. Treatment with P2X7 and A2A antagonists (AZ10606120 and SCH58261) markedly inhibited cancer growth and prevented tumor dissemination in an immune response-dependent manner. Finally, expression levels of P2X7, CD39, CD73, and A2A mRNAs were significantly higher in stage IV metastatic colon carcinoma patients. Furthermore, P2X7 and A2A expression increased in APC -mutated tumors and in spontaneous neoplasias within the colon mucosa of APC -mutated PIRC rats. Our study highlights the close interconnection between P2X7, A2A, CD39, and CD73 in colon carcinoma metastases. It identifies P2X7-dependent vesicle secretion as a new mechanism that favors metastatic dissemination and offers an innovative immunotherapeutic approach that targets vesicular release. Furthermore, we establish a first-time association between P2X7 and A2A overexpression and APC oncogene mutations, suggesting that these receptors could serve as potential biomarkers for advanced colon carcinoma. Biological sciences/Cancer/Metastasis Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer Biological sciences/Cell biology/Cell signalling/Ion channel signalling Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The tumor microenvironment (TME) is rich in extracellular ATP (eATP) and its hydrolytic derivative adenosine, which promotes pro and anti-tumor pathways acting on tumor cells and immune infiltrates ( 1 – 3 ). The best-characterized receptor for eATP in cancer is P2X7, which is expressed by both immune and tumor cells, promoting cancer growth ( 4 ), neovascularization ( 5 ), dissemination ( 6 ), and release of its ligand ATP ( 7 ), as well as activating tumor-eradicating immune responses ( 8 – 10 ). ATP and adenosine levels are strictly intertwined as, in the extracellular milieu, adenosine is produced from ATP by phosphates group loss mediated by the ectonucleotidases CD39 and CD73 ( 10 ). Interestingly, these enzymes and the primary adenosine receptor expressed in cancer (A2A) are emerging therapeutic targets in clinical trials designed to relieve immunosuppression and circumvent resistance to tumor immunotherapy ( 3 , 11 ). In addition, in the TME, A2A promotes VEGF secretion, tumor cell proliferation, and dissemination ( 12 , 13 ). Recent literature strongly supports the existence of crosstalk between the purinergic and adenosinergic systems in promoting cancer. For example, tumors growing in P2X7 null mice overexpress CD73, CD39, and A2A in either immune-infiltrating or tumor cells ( 7 , 14 ). Moreover, the mechanisms leading to the effectiveness of anti-CD39 antibodies as immune system reactivating drugs depend on P2X7 ( 15 , 16 ), highlighting the close link between key members of the purinergic system in the TME. All these findings prompted researchers to designate P2X7, CD39, CD73, and A2A as purinergic checkpoints ( 17 ). Extracellular vesicles, including exosomes and microvesicles, have been identified as cancer transformation and progression mediators, influencing metastatic dissemination ( 18 , 19 ). Cancer cells released from the primary tumor can engraft only in a favorable microenvironment known as the metastatic niche. The release of vesicles is a mechanism by which cancer cells influence the composition of distant extracellular microenvironments, causing the so-called metastatic niche preconditioning effect: reprogramming the secondary organ sites to favor tumor cell engraftment and, therefore, metastasis formation ( 20 ). However, although extracellular vesicle blockade holds promise as a therapeutic approach to prevent metastasis, no drugs able to interfere with this process are available on the market, probably because most of the targets identified so far are intracellular ( 21 ). The P2X7 receptor was long known for its ability to trigger the release of extracellular vesicles containing IL-1β, IL-18, and other cytokines from immune and nervous system cells ( 22 ). We have recently demonstrated that melanoma cells can also release exosomes and microvesicles upon P2X7 stimulation. These particles are characterized by miRNA content that is profoundly different from spontaneously released vesicles ( 6 ). Interestingly, vesicles released following P2X7 activation also contain ATP ( 6 , 23 , 24 ), thus suggesting that they might shape purinergic signaling in the TME. Indeed, it has been shown that extracellular vesicles can carry CD39 and CD73 and degrade ATP into immunosuppressive adenosine, supporting cancer growth ( 25 ). However, an in-depth investigation was missing linking the P2X7 /CD39/CD73 /A2A axis with extracellular vesicle release and the ability of these particles to influence ATP and adenosine in the TME to promote metastasis. Our study investigates P2X7-dependent vesicle secretion as a possible cause of colorectal cancer (CRC) metastasis and its antagonism as a new therapeutic approach to avoid cancer dissemination. CRC is one of the most common cancers and the second leading cause of cancer death worldwide. ( 26 , 27 ). The survival rates of patients with early-stage disease are increasing because of the efficacy of surgery, which is often combined with adjuvant therapy. However, late-stage metastatic CRC remains a clinical challenge ( 28 ). A bad prognosis due to metastasis is frequently due to mutations in the adenomatous polyposis coli ( APC ) gene associated with familiar forms of CRC but also often found in sporadic carcinomas ( 29 ). Although P2X7, CD39, CD73, and A2A have been separately associated with CRC development and progression ( 30 – 34 ) a systematic analysis of their crosstalk in metastatic spreading was missing. Results P2X7 blockade reduces the release of extracellular vesicles from colon carcinoma cells. We recently demonstrated the release of extracellular vesicles and exosomes following P2X7 stimulation in melanoma cells and how their miRNA content affects cancer cell migration ( 6 ). Therefore, we investigated whether a similar mechanism can be activated by the P2X7 receptor in colon carcinoma cells. At this aim, we selected CT26 and HCT-116 colon carcinoma cell lines that both express the P2X7 receptor (supplementary Fig. 1). Figure 1 shows that stimulation of P2X7 with its most potent agonist, BZ-ATP, causes the release of particles detectable by confocal microscopy in CT26 colon carcinoma cells (Fig. 1 A-B, see supplementary video 1). This phenomenon can also be activated in HCT-116 cells (see supplementary video 2). Interestingly, treatment with the P2X7 negative allosteric modulator AZ10606120 reduced particle release, as shown by confocal (Fig. 1 C-D, see supplementary video 3) and particle detection analysis (Fig. 1 E). Blockade with another receptor antagonist, A740003, which has a distinct chemical structure from AZ10606120, caused a comparable reduction in particle release, thus confirming the efficacy of receptor antagonism in decreasing vesicular release from cancer cells (Fig. 1 F). The mean diameter of the detected particles, measured using nanosight technology, was approximately 150–200 nm and did not vary upon P2X7 stimulation or antagonism (data not shown). Western Blot analysis of the vesicular content showed that particles released following ATP stimulation lost GM130 while gaining Alix staining compared to spontaneously released particles (Fig. 1 G), suggesting P2X7 activation-dependent changes in the vesicular nature and content. Interestingly, these particles also expressed P2X7, CD39, CD73, and A2A, indicating that they may regulate the purinergic/adenosinergic axis in the TME (Fig. 1 G). Indeed, particles not only carried ATP, as demonstrated by quinacrine staining (Fig. 1 A-D), but also caused an increase in pericellular ATP levels, which were more abundant when the vesicles were collected following P2X7 stimulation (Fig. 1 H, I). These measurements were performed using the pmeLUC probe, which allows the measurement of extracellular ATP on the plasma membrane of cancer cells expressing it ( 23 , 35 ). These data were confirmed by measuring free ATP in the supernatant using a classical luciferin/ luciferase assay (Fig. 1 J). Interestingly, adenosine levels in the cell supernatants were also increased by treatment with particles isolated following P2X7 stimulation (Fig. 1 K). In vivo administration of P2X7-released vesicles enhances colon carcinoma cell dissemination. To understand whether extracellular vesicles released upon P2X7 activation could play a role in colon carcinoma metastatic processes, we analyzed the effect of their administration on CT26 cell mobility with a scratch test assay (Fig. 2 A). These experiments showed that only particles released upon P2X7 stimulation (ATP-VS) and not those spontaneously released (S-VS) were able to increase colon carcinoma cell spreading in vitro and that administration of P2X7 agonists together with its antagonist prevented this effect (Fig. 2 A). Similar results have been obtained in vivo in a metastatic model obtained by injecting CT26 cells in the caudal vein of BALB/c syngeneic mice. Pretreatment of CT26 cells with vesicles released upon P2X7 activation significantly increased cancer cell spreading in the lungs (Fig. 2 B, C), systemic levels of the pro-inflammatory tumor-promoting cytokine IL-17 (Fig. 2 D), and metastasis formation (Fig. 2 E-H). Interestingly, receptor antagonism with AZ10606120 abrogated the vesicle-dependent dissemination to the lungs (Fig. 2 B, C, E, I) and IL-17 secretion (Fig. 2 D), strongly suggesting that P2X7 is a druggable target to prevent vesicle-dependent colon carcinoma metastatic progression. Interestingly, co-administration of microparticles released upon P2X7 stimulation not only increased cancer spreading (Fig. 2 A, B, C, E-I) but also tissue expression of both P2X7 (Fig. 3 A-E) and A2A (Fig. 3 F-J) and this phenomenon was reversible upon P2X7 blockade (Fig. 3 A, E, F J). P2X7 A2A combined antagonism reduces colon carcinoma growth, dissemination, and circulating IL-17 in a syngeneic mice model. To investigate the possible role of adenosine and the A2A receptor in P2X7-dependent metastatic dissemination, we tested the effects of P2X7 and A2A double blockade in vivo. P2X7 and A2A antagonism alone or in combination proved effective in reducing the spread of CT26 cells in our metastatic model (Fig. 4 A, B, D-I). P2X7 antagonist AZ10606120 and A2A antagonist SCH58261 were administered alone or in combination every three days (Fig. 4 ). Similar results were obtained when measuring the IL-17 circulating levels (Fig. 4 C). The significant reduction in metastatic spreading and engraftment observed with either P2X7 or A2A blockade suggests both receptors favoring CRC dissemination. Interestingly, when mimicking, with the same cells and mice strain, a primary CRC model obtained by subcutaneous injection, only co-administration of both drugs was effective in reducing cancer growth (Fig. 4 I, J) and the levels of IL-17 (Fig. 4 K). Moreover, only the combined antagonism of both receptors reduced the expression of P2X7 and A2A in the lungs of the metastasis-bearing mice (Fig. 5 A-J). Finally, when we performed similar experiments with HCT-116 human CRC cells in nude mice, we did not measure any effect of P2X7 and A2A blockade alone or in combination, either in subcutaneous or intravenous models (see supplementary Fig. 2), suggesting strong involvement of the immune response in the mechanism of action of the drugs. P2X7, CD39, CD73, and A2A are upregulated in metastatic colon carcinoma patients. To understand whether our findings on the role of the purinergic adenosinergic axis could also be translated to patients, we analyzed the expression of P2X7, CD39, CD73, and A2A in an array of 158 CRC specimens (Fig. 6 ). Our analysis showed that upregulation of both P2X7 human isoforms, P2X7A and P2X7B ( 36 , 37 ) are associated with stage IV CRC (Fig. 6 A, 6 C), a subset of patients characterized by a bad prognosis and metastatic dissemination. Similar results were obtained for CD39 (Fig. 6 E), CD73 (Fig. 6 G), and A2A (Fig. 6 I). In our tested samples, the mRNA that best increased according to the CRC stage was CD73 (Fig. 6 G). Interestingly, when we further analyzed data from stage IV CRC samples, subdividing them into those obtained from primary tumors versus metastatic specimens, we found upregulation of all purinergic checkpoints in secondary metastatic forms (Fig. 6 B, D, F, H, J). In these samples, we also reported a positive correlation between the expression levels of P2X7A and P2X7B (Spearman's coefficient 0,74), P2X7A and A2A (Spearman's coefficient 0,45), P2X7B and A2A (Spearman's coefficient 0,34) (Fig. 6 K). A moderate positive correlation between P2X7 and A2A was confirmed by the analysis of CRC samples from the Atlas database (Fig. 6 L). However, it was impossible to distinguish between P2X7A and B isoforms in this case. APC mutation in patient specimens and the PIRC rat correlates with P2X7 and A2A overexpression APC Oncogene mutational status is often associated with bad prognosis and metastatic dissemination in CRC patients. Therefore, to further corroborate our data, we also analyzed P2X7A, P2X7B, CD39, CD73, and A2A expression in mRNA extracted from human CRC samples coming from APC WT versus APC mutated tumors (Fig. 2 I-M). Interestingly, P2X7A, P2X7B, and A2A mRNA were increased in APC mutated tumors. In the same samples, the levels of CD39 and CD73 also showed a tendency to be upregulated in APC -mutated patients (Fig. 2 L-M). To substantiate the association between P2X7 and A2A to APC mutational status, we took advantage of a murine genetic model of intestinal tumorigenesis: PIRC rats carrying an APC mutation. These rats spontaneously develop tumors in the colon that faithfully reproduce familiar and sporadic CRC in humans ( 38 , 39 ). Immunohistochemistry demonstrated an upregulation of P2X7 and A2A in both the colon mucosa and colon tumors of PIRC rats as compared to the colon mucosa of WT rats (Fig. 2 A-H). Interestingly, this upregulation appeared to involve both cancer and immune cells (Fig. 2 E, H). In contrast, CD39 and CD73 levels were not altered in PIRC rats (see Additional Fig. 1 A-H). Discussion CRC still accounts for an high number of deaths worldwide ( 40 ). Despite recent advancements in survival expectations owing to emerging therapeutic options the metastatic forms of CRC remain a clinical challenge ( 41 , 42 ). These premises formed the basis of our study, which aimed to associate vesicular release triggered by eATP and the purinergic axis formed by P2X7/CD39/CD73 and A2A with CRC metastatic dissemination. Indeed, extracellular vesicles play important roles in preconditioning the metastatic niche, transforming it into a hospitable, tumor-friendly milieu that supports the engraftment and growth of cancer cells ( 43 ). eATP and its degradation product, adenosine, have been associated with CRC development, immune escape, and dissemination ( 10 , 30 , 44 ). However, an association between P2X7-dependent vesicular release and accumulation of ATP and adenosine in the TME leading to facilitation of metastasis was missing. Our study shows that P2X7 stimulation causes the release of extracellular vesicles from CRC cells (ATP-VS) that can promote metastasis formation in vivo (Figs. 5 , 6 ). Although we did not characterize ATP-VS in detail, we know that their content differs from that of particles spontaneously released from the same cells (S-VS) for the absence of GM 130 and the presence of Alix, suggesting that they are not apoptotic bodies and that they might include exosomes ( 45 ). P2X7 antagonists can revert ATP-mediated vesicular release, as demonstrated by confocal and nanosight experiments performed with receptor-blocking drugs of different chemical natures. Administration of ATP-VS increases both eATP and adenosine levels in CRC cell cultures. Interestingly, eATP concentration rises both in the vicinity of the plasmalemma, as measured by luciferase expressed in the outer layer of the plasma membrane (pmeLUC) ( 35 ), and in the supernatant of the cells. The increase in adenosine following ATP-VS administration was delayed compared with that of eATP, suggesting a hydrolytic origin. Indeed, ATP-VS carry CD39 and CD73, implying that they can enhance the production and accumulation of adenosine in the TME, as previously described in other oncologic contexts ( 25 ). We recently demonstrated that the ATP-VS released by melanoma cells and the miRNAs they carry favor cancer cell migration in vitro ( 6 ), and, we confirmed that a similar mechanism is activated in CRC. Moreover, we extended these findings to a live model of metastatic carcinogenesis, showing increased dissemination and engraftment in the lungs of CRC cells pre-treated with ATP-VS and intravenously injected in mice. More importantly, P2X7 antagonism can eliminate the metastatic advantage conferred by vesicles. The finding that blockade of P2X7-dependent vesicular release can prevent CRC dissemination is of particular relevance given that although inhibition of extracellular vesicle secretion holds promise for preventing tumor progression ( 46 ), no drugs that target vesicular secretion have been approved for human use ( 21 , 47 ). Our data show that vesicles released upon P2X7 activation were more effective than those spontaneously released by tumor cells, allowing CRC metastasis. This effect could be due to the fact that these particles carry prometastatic miRNAs ( 6 ), ATP ( 6 , 23 , 24 ), and mitochondria ( 24 , 48 ), which can activate the metabolic activity of cells ( 48 ) and shape the immune response ( 49 , 50 ). Moreover, the high concentrations of eATP present in the TME are compatible with continuous P2X7 activation, leading to vesicle release and even increase following most traditional oncological interventions, including chemotherapy and radiotherapy ( 11 ), suggesting that ATP-VS could favor the relapse of oncologic conditions following common treatments. Finally, several P2X7 antagonists have already been administered in patients with non-oncologic diseases, including Chron's, with little to no side effects ( 51 ). Therefore, they can be quickly transferred to a clinical setting to prevent vesicle-mediated metastasis. The levels of eATP and adenosine in the TME are interdependent because adenosine is generated from ATP via CD39 and CD73, which are also known to favor cancer growth through immune suppression. Recent evidence shows that the lack or inhibition of P2X7 alters eATP levels and modulates CD39, CD73, and adenosine receptor A2A in both immune-infiltrating and cancer cells ( 7 , 14 , 15 ) strongly suggesting that manipulation of P2X7 receptor activity affects the entire purinergic/adenosinergic axis in the TME. Indeed, we demonstrated that vesicles released upon P2X7 activation enhance both extracellular ATP and adenosine levels and that they carry not only P2X7 but also CD39, CD73, and A2A, suggesting that the ATP-VS influences the adenosinergic axis. Additionally, ATP-VS pretreatment of disseminating tumor cells enhanced the expression of both P2X7 and A2A receptors in tumors engrafting to the lungs. To explore whether the mechanisms driving P2X7-dependent metastasis also influenced the pro-tumoral adenosinergic pathway, we investigated the effects of concurrent blockade of P2X7 and A2A receptors in vivo . P2X7 and A2A antagonists, alone or in combination, were able to reduce the metastatic spread of CT26 CRC cells in a syngeneic mouse model. Thus confirming the importance of both receptors in the promotion of CRC metastasis. Notably, double antagonism, although not causing any evident extra reduction in metastatic spreading, was the only treatment able to decrease the expression of both P2X7 and A2A in the engrafted tumors. Thus suggesting a more efficacious action in the downmodulation of purinergic checkpoints and hinting at an addictive effect of the double antagonism on the growth of metastases following engraftment. The administration of P2X7 or A2A antagonists alone was not sufficient to mitigate subcutaneous primary tumor growth, which was nonetheless significantly reduced by combined anti-P2X7/A2A blockade. Interestingly, none of the antagonists alone or in combination was able to reduce CRC growth in the nude mouse model devoid of T cell-mediated responses, implicating a central role of the immune system in their mechanism of action. In fact, in the fully immune-competent syngeneic model, circulating levels of IL-17 were also reduced by P2X7 and A2A antagonism, following a pattern similar to that observed for tumor growth and metastatic dissemination. IL-17 is a cytokine implicated in autoimmune diseases and the maintenance of colon homeostasis ( 52 , 53 ), which promotes CRC development, progression, and metastasis and reduces the efficacy of immune therapy ( 53 , 54 ). Both P2X7 and A2A have been previously associated with Th17 cells and IL-17 secretion in different pathological contexts ( 14 , 55 – 57 ). However, to our knowledge, this is the first demonstration that blocking P2X7 and A2A, alone or in combination, can significantly reduce the levels of IL-17 in CRC murine models. Interestingly, in the metastatic context, IL-17 levels can also be upregulated by treatment with vesicles derived from CRC cells stimulated with the P2X7 agonist ATP, suggesting that the cargo of these vesicles comprising ATP and miRNAs ( 6 , 24 ) plays a central role in modulating Th17 pathways. To corroborate our data in a patient cohort we demonstrated that the mRNAs levels of the two main P2X7 human isoforms, P2X7A and P2X7B, were upregulated in patients with stage IV CRC. Moreover, they almost doubled when comparing expression in primary stage IV tumors versus metastatic forms. These data confirm the association of both isoforms with the metastatic transformation of other tumors, such as neuroblastoma ( 5 ), melanoma ( 6 ), osteosarcoma ( 58 ), and prostate cancer ( 59 ). Similar results were obtained for CD39, CD73, and A2A expression with CD73, which was also upregulated in stage III CRC samples. When performing a correlation analysis in metastatic specimens, P2X7A and P2X7B were strongly associated; however, there was also a moderate correlation between both isoforms and A2A. This correlation was confirmed by analysis of the Atlas database, which, unfortunately, does not allow for the distinction between P2X7 isoforms. In subsequent experiments, we investigated the correlation between the upregulation of P2X7 and A2A receptors and APC oncogene mutational status. Our findings revealed that mRNA expression of both P2X7A and P2X7B isoforms, as well as A2A, was significantly elevated in APC -mutated colorectal cancer patient samples compared to wild-type controls. Similar trends were observed in tumors spontaneously developing in the colonic mucosa of APC -mutated PIRC rats, showing upregulation of both receptors in mucosal, cancerous and immune cells. In contrast, the expression levels of CD39 and CD73 remained relatively unchanged in both the PIRC rat model and APC -mutated patients. To our knowledge, this is the first demonstration of P2X7 upregulation in a tumor that spontaneously develops in an oncogene-mutated murine model and the first association in cancer patients between P2X7 isoforms and APC mutations. APC mutations were recently correlated with a poor response to immunotherapy in CRC ( 60 ), and given the role of both receptors in the cancer immune response ( 8 , 11 ), it is tempting to speculate that the upregulation of P2X7 and A2A in these patients could be partly responsible for this phenomenon. Moreover, although the number of APC -mutated patients we tested was limited, we noticed that those were more frequently characterized by metastatic spread in organs other than the liver (supplementary Table 1) and, therefore, more challenging to treat, were also those overexpressing P2X7 and A2A suggesting that a P2X7-A2A targeting therapy might be indicated to prevent metastatic spreading in this patient population. In conclusion, this study elucidates a novel mechanism by which vesicular release modulates ATP and adenosine levels in cancer, thereby promoting metastatic dissemination. Our findings present an exciting therapeutic opportunity to target tumoral vesicular release, potentially enhancing immune responses against cancer. This innovative approach could significantly advance the field of theranostics by providing new strategies for combating metastasis and improving patient outcomes. Materials/Subjects and Methods Cell cultures and transfection CT26 murine and HCT116 human colon carcinoma cell lines (EP-CL-0071, EP-CL-0096, CliniScience, Amsterdam, Netherlands) were cultured in RPMI 1640 (Carlo Erba Reagents, Milan, Italy) and McCoy's 5A (Euroclone, Milan, Italy) medium, respectively. Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Euroclone). CT26 and HCT116 cells were stably transfected with intracellular Luc 2 and plasma membrane pmeLUC luciferase probes using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Stably transfected cells were maintained with hygromycin B (0.2 mg/mL, Roche, Basel, Switzerland ) or geneticin (0.4 mg/mL, Sigma-Aldrich, Darmstadt, Germany). All cells were routinely tested using the Mycoplasma kit detection from Applied Biological Material (Richmond, Canada). Confocal microscopy CT26 and HCT116 cells were seeded onto 24 mm glass coverslips (Thermo Fisher Scientific), and loaded with 1µM plasma membrane dye PKH26GL (Sigma-Aldrich) or FM4-64 (Thermo Fischer Scientific) plus 1µM Quinacrine dihydrochloride (Q3251, Sigma Aldrich) for ATP and nucleic acid staining. Live single-cell imaging was performed with an Olympus FV3000 confocal microscope using the FV31S-SW software (Olympus, Tokyo, Japan). The excitation wavelengths were 445nm and 594 nm, respectively. Emission was measured in the 610–710 nm range for red staining and in the 460–500 nm range for green staining. Vesicles concentration Vesicles were concentrated from the cell culture supernatant by centrifugation. 10 x 10 5 cells were incubated in saline solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO 4, 1 mM NaH 2 PO 4 , 20 mM HEPES, 5.5 mM glucose, 5 mM NaHCO 3 , 1 mM CaCl 2 , pH 7.4) with or without 3mM ATP for 30 minutes at 37°C with 5% CO 2, and in some conditions, pre-treated for 10 minutes with the P2X7 antagonists AZ10606120 (5 µM) (Tocris Bioscience, Bristol, UK) or A740003 (20 µM) (Tocris Bioscience). The supernatant was first depleted of cells and debris by centrifugation at 2000 x g for 10 minutes at 4°C. The vesicles were then pelleted at 20000 x g for one hour at 4°C. ATP and adenosine measure 2 x 10 4 CT26 pmeLUC cells were seeded in a 96-well plate and used as the ATP sensors. Spontaneously released (S-VS) or ATP stimulated (ATP-VS) vesicles were collected from CT26 cells as described above and resuspended in 10 µL of PBS at a concentration of 3 µg/µl. D- luciferin (Promega, Madison, Wisconsis, USA) was added to the wells containing CT26 pmeLUC at a concentration of 60 µg/mL. Basal luminescence emission was measured for 5 minutes using an IVIS Lumina Luminometer (Perkin Elmer, Waltham, Massachusetts, USA). Vesicles or 10 µL of PBS as a control were added to the cells, and luminescence emission was acquired for additional 5 minutes. Photon emission was quantified using the Living Image® Software (Perkin Elmer) as total photons/seconds (p/s). Changes in ATP concentration were expressed as a fold increase on basal luminescence emission. ATP and adenosine levels were measured in the supernatant of CT26 cells 30 and 90 minutes after the addition of vehicle (CTR), S-VS, or ATP-VS using ENLITEN rLuciferin/Luciferase reagent (Promega) ( 7 ) or adenosine assay kit (Cell Biolabs, Inc. MET-5090, San Diego, California, USA). Western Blot S-VS and ATP-VS were collected in RIPA buffer plus Halt™ Protease and Phosphatase inhibitor cocktail EDTA-free 100 x (Sigma-Aldrich). Protein lysates were loaded and separated on 4–12% NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane, and incubated overnight with the following primary antibodies: anti-GM130 1:500 (Exosomal Marker Antibody Sample Kit, Cell Signaling Technology), anti-Alix 1:500 (Exosomal Marker Antibody Sample Kit, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-P2X7 1:300 (P8232, Sigma Aldrich) anti-CD73 1:250 (Bioss Antibodies, Woburn, Massachusetts, USA ), anti-CD39 1:500 (Cohesion Bioscience, London, UK) anti-A2A (SC32261, Santa Cruz Biotechnology, Dallas, Texas, USA). Incubation with secondary anti-rabbit (Thermo Fisher Scientific) or anti-mouse (Thermo Fisher Scientific) antibodies (1:2000) was performed for 1 hour. Protein bands were visualized using the ECL HRP Chemiluminescent Substrate ETA C ULTRA 2.0 (Cyanagen Srl, Bologna, Italy) with a Licor C-Digit Model 3600. Expression of P2X7 and A2A in cell lysates from CT26-Luc2 and HCT116-Luc2 cells was demonstrated by immunoblot as previously described.( 14 ). Nanoparticle tracking analysis Vesicles were resuspended in 60 µL of filtered PBS and diluted 1:100 or 1:500 in PBS. Vesicles were tracked using the Nanosight system (Nanosight LM10, Malvern, UK), according to the manufacturer's instructions. Data were analyzed using the NanoSight Software NTA 3.2 Dev Build 3.2.16. Scratch recovery assay A Wound Healing Assay (ab242285, Abcam, Cambridge, UK) was used for scratch tests in serum-free RPMI 1640 medium. CT26 cells were seeded in 24-well plates containing inserts. When they reached confluence, the insert was removed to generate a 0.9 mm wound field. Cells were treated with 10 µl of PBS (control), S-VS, ATP-VS, or ATP-VS collected following pretreatment with the P2X7 antagonist AZ10606120. Pictures of scratches were acquired at time 0 (T0) and after 24 and 48 hours (T24, T48) with a phase-contrast optical DM IL Led Leica Microsystem microscope (LEICA ICC50 HD, Wetzlar, Germany). ImageJ Software was used for scratch measurement. The percentage closure was calculated by comparison with T0. Murine models A syngeneic experimental metastasis model was established by injecting 2.5 x 10 5 CT26 Luc2 cells into the tail vein of BALB/c 4–6 weeks-old female mice (Envigo, Udine, Italy). Mice were randomized into four groups of 6 animals each, and the operator was blinded to the allocation group. In a first set of experiments CT26 Luc2 cells were intravenously injected with vesicles collected as described in the "Vesicles concentration" paragraph. Cell dissemination was monitored every 72 hours by measuring luciferase Luc2 photon emission using an IVIS Lumina Luminometer (Perkin Elmer) as described previously ( 6 ). In a second set of experiments, animals were treated every 72 hours with an i.p. injection of placebo (PBS + 0.002% DMSO), the P2X7 antagonist AZ10606120 (2 mg/Kg) (Tocris Bioscience), the A2A antagonist SCH58261 (1 mg/Kg) (Tocris Bioscience), and a combination of both antagonists. The same antagonist administration schedule was also employed in a xenotransplant model obtained by tail vein injection of 2 x 10 6 HCT-116-Luc2 cells in nude/nude mice (Envigo). Mice were randomized into four groups of 7 animals each, and the operator was blinded to the allocation group. To mimic primary colon carcinoma, 5 x 10 5 CT26 cells were subcutaneously injected into 6-week-old BALB/c female mice (Envigo). The mice were randomized into four groups of 12 animals, and the operator was blinded to the allocation group. After five days, animals were treated every 72 hours with an intraperitoneal (i.p.) injection of placebo (PBS + 0.002% DMSO), the P2X7 antagonist AZ10606120 (2 mg/Kg) (Tocris Bioscience), the A2A antagonist SCH58261 (1 mg/Kg) (Tocris Bioscience), and a combination of both antagonists. Tumor size was determined as described in ( 7 ). The same antagonist administration schedule was also employed in a xenotransplant model obtained by subcutaneous injection of 2 x 10 6 HCT-116-Luc2 cells in nude/nude mice (Envigo). Mice were randomized into four groups of 6 animals each, and the operator was blinded to the allocation group. Blood samples were collected and prepared as previously described ( 7 ). All animal procedures were approved by the University of Ferrara Ethics Committee and the Italian Ministry of Health (Italian D. Lgs 26/204) and were in accordance with generally accepted guidelines for the welfare and use of animals in cancer research ( 61 ). Tissue slides from the colons of WT and PIRC rats and related tumors were obtained from archived materials available at Caderni's laboratory ( 38 , 39 ). Cytokine quantification Plasma levels of interleukin 17 were measured using the Simple Plex™ Cartridge Kit with Ella Automated Immunoassay System (Biotechne, Minneapolis, Minnesota, USA). Histology Tissue slides from mouse lungs were processed as previously described ( 4 ). Images were acquired using the NIS-Element Software with a Nikon Eclipse H550L microscope (Nikon Europe, Amstelveen, Netherlands). Metastasis formation in the lungs was analyzed by quantifying the area of the samples covered by clearly distinguishable tumor cells that were visible by hematoxylin/eosin staining. The total area of metastasis is expressed as a percentage of the entire lung area. The areas were quantified using ImageJ Software. Immunohistochemistry Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology). The protocol used for tissue staining has been described previously ( 14 ). Images were acquired using a Nikon Eclipse H550L microscope and the NIS-Element Software (Nikon). The percentage of positive cells was quantified using QuPath open-source software. ( 62 ). Quantitative Real-Time PCR. Samples from human colon carcinoma patients were obtained from five commercial arrays (TissueScan™ cDNA arrays HCRT301, HCRT302, HCRT303, HCRT304, HCRT305, OriGene, Rockville, Maryland, USA). Samples in which mRNA levels for housekeeping genes were not detectable were excluded from the analysis. A total of 158 patients were analyzed and subdivided according to the diagnostic phase into stage I (n = 24), stage II (n = 50), stage III (n = 52) and stage IV (n = 32). Stage IV patient data were further analyzed according to the origin of the cDNA from the primary (n = 21) or metastatic (n = 11) specimens. CRC samples cDNA were used as a template for quantitative Real-Time PCR (qRT-PCR) using TaqMan®MGB probes, FAM™ dye-labeled (20X), and TaqMan Gene Expression Master Mix 2X (Applied Biosystems, Waltham, Massachusetts, USA). Taqman custom probes and primers for P2X7A and P2X7B were previously described by Pegoraro et al. ( 63 ). Taqman predesigned probes for the other genes were, respectively: Hs00969556_m1 ENTPD1 for CD39, Hs00159686_m1 NT5E for CD73, and Hs00169123_m1 ADORA2A for A2A (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1, Applied Biosystems) was used as a housekeeping gene. qRT-PCR was performed using an AB PRISM 7300 Step One Real-Time PCR system (Applied Biosystems) as previously described ( 6 ). Formalin-fixed paraffin-embedded tumor samples (n = 12) from patients with colon carcinoma were collected according to a protocol approved by the Istituto Romagnolo per lo studio dei tumori (IRST) Ethics Committee (CEROM IRST IRCCS-AVR, protocol code: IRST B125 approved on the 19th of March 2021). All patients signed an informed consent form before surgery. No specific inclusion or exclusion criteria were set. Patients' information is summarized in supplementary table 1 . Patients were stratified according to APC mutational status evaluated by whole exome sequencing ( APC variants were considered with a variant allele frequency of 5% or greater). Total RNA was extracted from paraffin-embedded tissue sections using the Maxwell RSC RNA FFPE Kit (Promega, cat. no. AS1440) on a Maxwell CSC 48 automated nucleic acid extraction system (Promega) according to manufacturer instructions. Total RNA was quantified using the Qubit RNA High Sensitivity (HS) Assay Kit (Invitrogen, cat no. Q32852). 200 ng of total RNA were reverse-transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, cat. no Q32852). Real-time PCR was performed using the 7500 Real-time PCR System (Applied Biosystem, USA) as previously described ( 64 ). Gene expression correlation Spearman's correlation analysis among the genes tested in our stage IV metastatic patients' commercial datasets was performed using GraphPad Prism Software (GraphPad, La Jolla, California, USA). Moreover, P2X7 and A2A gene expression was evaluated in colon adenocarcinoma samples from the Cancer Genome Atlas database (TGCA) using the GEPIA web server ( 65 ). Spearman's correlation coefficient was applied for the analysis calculation with a non-log scale, whereas the data were visualized using the log-scale axis. Statistics All data are shown as mean ± standard error of the mean (SEM). Significance was calculated assuming equal standard deviation and variance, with a two-tailed Student's t-test or ordinary one-way ANOVA performed using GraphPad Prism Software (GraphPad). For each in vivo experiment, the group size and statistical power were selected following computation a priori , based on previous data ( 6 , 7 , 63 ), using an online sample size calculator (clincalc.com/stats/simplesize.aspx). Statistical significance was set at P-values lower than 0.05. Declarations Acknowledgments The authors thank Prof. Anna Lisa Giuliani, Dr. Simonetta Falzoni, Dr. Mario Tarantini and Prof. Massimo Bonora for fruitful discussion, Mrs. Marzia Scarletti and Dr. Federica Poletti for technical assistance. This article is based upon work from PRESTO COST Action CA21130, supported by COST (European Cooperation in Science and Technology) www.cost.eu; www.p2xcost.eu. Conflict of Interest Statement The late FDV was a member of the Scientific Advisory Board of Biosceptre Ltd. (UK), and a consultant at Breye Therapeutics (Denmark), and at Crosslink Therapeutics Inc (USA). All other authors declare no conflict of interest. Author Contribution Statement EA and AP conceived and designed the study and wrote, reviewed, and revised the manuscript. AP, EDM, LR, MZ, SC, LA, MG, and EA performed the experiments. AP, EDM, LR, MZ, SC, GC, PU, and EA analyzed and interpreted the data. MZ, PU, AP, GG collected and processed patients' samples. FDV and LA participated in the experimental design. All authors reviewed and approved the final manuscript. Ethics Statement Patients' samples were collected in accordance with the IRST Ethics committee (CEROM IRST IRCCS-AVR, protocol code: IRST B125 approved on the 19th of March 2021). All the patients had signed informed consent prior to surgery. The animal study was reviewed and approved by Organismo Preposto al Benessere Animale (OPBA, organism for Animal Wellbeing), University of Ferrara and Italian Ministry of Health. Funding Statement This work was supported by the Italian Association for Cancer Research (AIRC) Grants to EA (IG22837), the PUR-THER, TRANSCAN3 Project to EA. Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 20225LKPYA to EA and LA). Data Availability Statement The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Di Virgilio F, Sarti AC, Falzoni S, De Marchi E, Adinolfi E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat Rev Cancer. 2018;18(10):601-18. Chiarella AM, Ryu YK, Manji GA, Rustgi AK. Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment. Trends Cancer. 2021;7(8):731-50. Bai X, Li Q, Peng X, Li X, Qiao C, Tang Y, et al. P2X7 receptor promotes migration and invasion of non-small cell lung cancer A549 cells through the PI3K/Akt pathways. Purinergic Signal. 2023. Adinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, et al. Expression of P2X7 receptor increases in vivo tumor growth. Cancer Res. 2012;72(12):2957-69. Amoroso F, Capece M, Rotondo A, Cangelosi D, Ferracin M, Franceschini A, et al. The P2X7 receptor is a key modulator of the PI3K/GSK3beta/VEGF signaling network: evidence in experimental neuroblastoma. Oncogene. 2015;34(41):5240-51. Pegoraro A, De Marchi E, Ferracin M, Orioli E, Zanoni M, Bassi C, et al. P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells. Cell Death Dis. 2021;12(12):1088. De Marchi E, Orioli E, Pegoraro A, Sangaletti S, Portararo P, Curti A, et al. The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment. Oncogene. 2019;38(19):3636-50. Adinolfi E, De Marchi E, Orioli E, Pegoraro A, Di Virgilio F. Role of the P2X7 receptor in tumor-associated inflammation. Curr Opin Pharmacol. 2019;47:59-64. Kepp O, Bezu L, Yamazaki T, Di Virgilio F, Smyth MJ, Kroemer G, et al. ATP and cancer immunosurveillance. EMBO J. 2021;40(13):e108130. Yegutkin GG, Boison D. ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain. Pharmacol Rev. 2022;74(3):797-822. Zanoni M, Pegoraro A, Adinolfi E, De Marchi E. Emerging roles of purinergic signaling in anti-cancer therapy resistance. Front Cell Dev Biol. 2022;10:1006384. Merighi S, Battistello E, Giacomelli L, Varani K, Vincenzi F, Borea PA, et al. Targeting A3 and A2A adenosine receptors in the fight against cancer. Expert Opin Ther Targets. 2019;23(8):669-78. de Araujo JB, Kerkhoff VV, de Oliveira Maciel SFV, de Resende ESDT. Targeting the purinergic pathway in breast cancer and its therapeutic applications. Purinergic Signal. 2021;17(2):179-200. De Marchi E, Pegoraro A, Turiello R, Di Virgilio F, Morello S, Adinolfi E. A2A Receptor Contributes to Tumor Progression in P2X7 Null Mice. Front Cell Dev Biol. 2022;10:876510. Yan J, Li XY, Roman Aguilera A, Xiao C, Jacoberger-Foissac C, Nowlan B, et al. Control of Metastases via Myeloid CD39 and NK Cell Effector Function. Cancer Immunol Res. 2020;8(3):356-67. Casey M, Segawa K, Law SC, Sabdia MB, Nowlan B, Salik B, et al. Inhibition of CD39 unleashes macrophage antibody-dependent cellular phagocytosis against B-cell lymphoma. Leukemia. 2023;37(2):379-87. Demeules M, Scarpitta A, Hardet R, Gonde H, Abad C, Blandin M, et al. Evaluation of nanobody-based biologics targeting purinergic checkpoints in tumor models in vivo. Front Immunol. 2022;13:1012534. Sohal IS, Kasinski AL. Emerging diversity in extracellular vesicles and their roles in cancer. Front Oncol. 2023;13:1167717. Jeppesen DK, Zhang Q, Franklin JL, Coffey RJ. Extracellular vesicles and nanoparticles: emerging complexities. Trends Cell Biol. 2023;33(8):667-81. Becker A, Thakur BK, Weiss JM, Kim HS, Peinado H, Lyden D. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell. 2016;30(6):836-48. Catalano M, O'Driscoll L. Inhibiting extracellular vesicles formation and release: a review of EV inhibitors. J Extracell Vesicles. 2020;9(1):1703244. Lombardi M, Gabrielli M, Adinolfi E, Verderio C. Role of ATP in Extracellular Vesicle Biogenesis and Dynamics. Front Pharmacol. 2021;12:654023. D'Arrigo G, Gabrielli M, Scaroni F, Swuec P, Amin L, Pegoraro A, et al. Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein. J Extracell Vesicles. 2021;10(9):e12114. Vultaggio-Poma V, Falzoni S, Chiozzi P, Sarti AC, Adinolfi E, Giuliani AL, et al. Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation. Theranostics. 2022;12(2):859-74. Carotti V, Rigalli JP, van Asbeck-van der Wijst J, Hoenderop JGJ. Interplay between purinergic signalling and extracellular vesicles in health and disease. Biochem Pharmacol. 2022;203:115192. Sedlak JC, Yilmaz OH, Roper J. Metabolism and Colorectal Cancer. Annu Rev Pathol. 2023;18:467-92. Waldum H, Fossmark R. Inflammation and Digestive Cancer. Int J Mol Sci. 2023;24(17). Shin AE, Giancotti FG, Rustgi AK. Metastatic colorectal cancer: mechanisms and emerging therapeutics. Trends Pharmacol Sci. 2023;44(4):222-36. Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330-7. Kunzli BM, Bernlochner MI, Rath S, Kaser S, Csizmadia E, Enjyoji K, et al. Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer. Purinergic Signal. 2011;7(2):231-41. Calik I, Calik M, Turken G, Ozercan IH. A promising independent prognostic biomarker in colorectal cancer: P2X7 receptor. Int J Clin Exp Pathol. 2020;13(2):107-21. Feng Y, Xu X, Zhang J, Sanderson C, Xia J, Bu Z, et al. CD39(+) tumor infiltrating T cells from colorectal cancers exhibit dysfunctional phenotype. Am J Cancer Res. 2024;14(2):585-600. Messaoudi N, Cousineau I, Arslanian E, Henault D, Stephen D, Vandenbroucke-Menu F, et al. Prognostic value of CD73 expression in resected colorectal cancer liver metastasis. Oncoimmunology. 2020;9(1):1746138. Ye H, Zhao J, Xu X, Zhang D, Shen H, Wang S. Role of adenosine A2a receptor in cancers and autoimmune diseases. Immun Inflamm Dis. 2023;11(4):e826. De Marchi E, Orioli E, Pegoraro A, Adinolfi E, Di Virgilio F. Detection of Extracellular ATP in the Tumor Microenvironment, Using the pmeLUC Biosensor. Methods Mol Biol. 2020;2041:183-95. Pegoraro A, De Marchi E, Adinolfi E. P2X7 Variants in Oncogenesis. Cells. 2021;10(1). Adinolfi E, De Marchi E, Grignolo M, Szymczak B, Pegoraro A. The P2X7 Receptor in Oncogenesis and Metastatic Dissemination: New Insights on Vesicular Release and Adenosinergic Crosstalk. Int J Mol Sci. 2023;24(18). Femia AP, Soares PV, Luceri C, Lodovici M, Giannini A, Caderni G. Sulindac, 3,3'-diindolylmethane and curcumin reduce carcinogenesis in the Pirc rat, an Apc-driven model of colon carcinogenesis. BMC Cancer. 2015;15:611. Vitali F, Tortora K, Di Paola M, Bartolucci G, Menicatti M, De Filippo C, et al. Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers. Sci Rep. 2022;12(1):1432. Cervantes A, Adam R, Rosello S, Arnold D, Normanno N, Taieb J, et al. Metastatic colorectal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2023;34(1):10-32. Zeineddine FA, Zeineddine MA, Yousef A, Gu Y, Chowdhury S, Dasari A, et al. Survival improvement for patients with metastatic colorectal cancer over twenty years. NPJ Precis Oncol. 2023;7(1):16. Bekaii-Saab TS, Barzi A, Cusnir M. Improving survival in metastatic colorectal cancer through optimized patient selection. Clin Adv Hematol Oncol. 2024;22 Suppl 4(5):1-20. Urabe F, Patil K, Ramm GA, Ochiya T, Soekmadji C. Extracellular vesicles in the development of organ-specific metastasis. J Extracell Vesicles. 2021;10(9):e12125. D'Antongiovanni V, Fornai M, Pellegrini C, Benvenuti L, Blandizzi C, Antonioli L. The Adenosine System at the Crossroads of Intestinal Inflammation and Neoplasia. Int J Mol Sci. 2020;21(14). Welsh JA, Goberdhan DCI, O'Driscoll L, Buzas EI, Blenkiron C, Bussolati B, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13(2):e12404. Maacha S, Bhat AA, Jimenez L, Raza A, Haris M, Uddin S, et al. Extracellular vesicles-mediated intercellular communication: roles in the tumor microenvironment and anti-cancer drug resistance. Mol Cancer. 2019;18(1):55. Irep N, Inci K, Tokgun PE, Tokgun O. Exosome inhibition improves response to first-line therapy in small cell lung cancer. J Cell Mol Med. 2024;28(4):e18138. Falzoni S, Vultaggio-Poma V, Chiozzi P, Tarantini M, Adinolfi E, Boldrini P, et al. The P2X7 Receptor is a Master Regulator of Microparticle and Mitochondria Exchange in Mouse Microglia. Function (Oxf). 2024;5(4). Pizzirani C, Ferrari D, Chiozzi P, Adinolfi E, Sandona D, Savaglio E, et al. Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells. Blood. 2007;109(9):3856-64. Longo Y, Mascaraque SM, Andreacchio G, Werner J, Katahira I, De Marchi E, et al. The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation. Front Immunol. 2024;15:1360140. Iqbal J, Bano S, Khan IA, Huang Q. A patent review of P2X7 receptor antagonists to treat inflammatory diseases (2018-present). Expert Opin Ther Pat. 2024;34(4):263-71. Huangfu L, Li R, Huang Y, Wang S. The IL-17 family in diseases: from bench to bedside. Signal Transduct Target Ther. 2023;8(1):402. Razi S, Baradaran Noveiry B, Keshavarz-Fathi M, Rezaei N. IL-17 and colorectal cancer: From carcinogenesis to treatment. Cytokine. 2019;116:7-12. Liu C, Liu R, Wang B, Lian J, Yao Y, Sun H, et al. Blocking IL-17A enhances tumor response to anti-PD-1 immunotherapy in microsatellite stable colorectal cancer. J Immunother Cancer. 2021;9(1). D'Addio F, Vergani A, Potena L, Maestroni A, Usuelli V, Ben Nasr M, et al. P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes. J Clin Invest. 2018;128(8):3490-503. Tokano M, Matsushita S, Takagi R, Yamamoto T, Kawano M. Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor. Brain Behav Immun Health. 2022;26:100544. Wang L, Wan H, Tang W, Ni Y, Hou X, Pan L, et al. Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17 cells in allergic asthma. Clin Respir J. 2018;12(1):149-57. Tattersall L, Shah KM, Lath DL, Singh A, Down JM, De Marchi E, et al. The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties. J Bone Oncol. 2021;31:100398. Song H, Arredondo Carrera HM, Sprules A, Ji Y, Zhang T, He J, et al. C-terminal variants of the P2X7 receptor are associated with prostate cancer progression and bone metastasis - evidence from clinical and pre-clinical data. Cancer Commun (Lond). 2023;43(3):400-4. Li B, Zhang G, Xu X. APC mutation correlated with poor response of immunotherapy in colon cancer. BMC Gastroenterol. 2023;23(1):95. Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. Guidelines for the welfare and use of animals in cancer research. Br J Cancer. 2010;102(11):1555-77. Bankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Sci Rep. 2017;7(1):16878. Pegoraro A, Orioli E, De Marchi E, Salvestrini V, Milani A, Di Virgilio F, et al. Differential sensitivity of acute myeloid leukemia cells to daunorubicin depends on P2X7A versus P2X7B receptor expression. Cell Death Dis. 2020;11(10):876. Zanoni M, Sarti AC, Zamagni A, Cortesi M, Pignatta S, Arienti C, et al. Irradiation causes senescence, ATP release, and P2X7 receptor isoform switch in glioblastoma. Cell Death Dis. 2022;13(1):80. Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45(W1):W98-W102. 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Ferrara","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"Di","lastName":"Virgilio","suffix":""}],"badges":[],"createdAt":"2024-10-18 07:52:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5287461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5287461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07897-2","type":"published","date":"2025-08-04T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68203821,"identity":"783a0f65-7fcc-4f01-910c-3a94f3cef50e","added_by":"auto","created_at":"2024-11-04 16:01:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1942100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColon carcinoma cells release vesicles upon P2X7 activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of CT26 cells untreated (A), CT26 cells stimulated with the P2X7 agonist BzATP 300 µM (B), CT26 cells treated with AZ10606120 5 µM (C), and CT26 cells treated with AZ10606120 5 µM and stimulated with BzATP (D) were acquired using confocal microscopy. The cell membrane was stained with the red fluorescent dye PKH26GL and the nucleic acid content was stained with the green fluorescent dye quinacrine. (C) Concentration of vesicles released spontaneously (S-VS), after stimulation of P2X7 with 3 mM ATP (ATP-VS), and after stimulation of P2X7 from cells treated with antagonist AZ10606120 (AZ10606120+ATP-VS) (n= 5). (F) Concentration of S-VS, ATP-VS, and after stimulation of P2X7 from cells treated with the antagonist A740003B (A74003 + ATP-VS) (n= 5). (G) Western blot for GM130, Alix, P2X7, CD39, CD73, and A2A in S-VS and ATP-VS. Changes in photon emission (H) of CT26 pmeLUC after 5 minutes of exposure to the vehicle (CTR), S-VS, and ATP-VS from CT26 cells (n=4). Quantification of luminescence changes was expressed as a fold increase on time 0. Representative images of photon emissions using the pmeLUC sensor (I). Changes in ATP (J) and adenosine (K) concentrations in the supernatants of CT26 cells treated with CTR, S-VS, and ATP-VS (n=3). The concentration was expressed as a fold increase on time 0. *p\u0026lt; 0.05, **p\u0026lt;0.001, ****p\u0026lt; 0.00001\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/23d3e7c78a1f9935f66f94ba.png"},{"id":68204921,"identity":"faa540a2-10cd-4161-91a8-e07533bc7462","added_by":"auto","created_at":"2024-11-04 16:09:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2308246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVesicles released from CRC cells upon P2X7 stimulation increase cell dissemination and metastasis formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantification of the migration capacity of CT26 cells alone or incubated with S-VS, ATP-VS, and AZ10606120 + ATP-VS. The migration ability is expressed as the percentage of the area evaluated after 24 and 48 hours from the scratch formation (n=3 per condition). (B) Photon emission of mice intravenously injected with only CT26 Luc2 cells or CT26 Luc2 pre-treated with S-VS, ATP-VS, and AZ10606120 + ATP-VS at days 11 and 14 (n=8 per condition). (C) Images of CT26 Luc2 cells' luminescence emission were captured thanks to a luminometer for small animals on day 14. (D) Systemic level of the pro-inflammatory cytokine IL-17 in mice injected intravenously with only CT26 Luc2 cells or CT26 Luc2 pre-treated with S-VS, ATP-VS, and AZ10606120 + ATP-VS (n=8 per condition). (E) Area of lung metastasis in mice injected intravenously with only CT26 Luc2 cells or CT26 Luc2 pre-treated with S-VS, ATP-VS, and AZ10606120 + ATP-VS expressed as a percentage of total lung area (n=3 per condition). Representative hematoxylin/eosin staining of lungs from mice injected with CT26Luc2 cells (F), CT26Luc2 cells + S-VS (G), CT26Luc2 cells + ATP-VS (H), CT26Luc2 cells + AZ10606120 + ATP-VS (I).\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/56a4138092badac472aab140.png"},{"id":68203826,"identity":"c72ecc20-7128-4071-a859-8d1e6af2cd59","added_by":"auto","created_at":"2024-11-04 16:01:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2591716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVesicles released from CRC cells upon P2X7 stimulation increase the expression of P2X7 and A2A in the metastatic milieu.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePercentage of lung cells positive to P2X7 (A) and A2A (F) staining in mice injected intravenously with only CT26 Luc2 cells or CT26 Luc2 pre-treated with S-VS, ATP-VS, and AZ10606120 + ATP-VS. Representative immunohistochemistry images of mice injected intravenously with only CT26 Luc2 cells (B, G) or CT26 Luc2 pre-treated with S-VS (C, H), ATP-VS (D, I), and AZ10606120 + ATP-VS (E, J). *p\u0026lt; 0.05, **p\u0026lt;0.001, ***p\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/f29ed891056bdbb3b785a030.png"},{"id":68203832,"identity":"722ea8cd-f780-404c-850f-3c458efb5a18","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2134679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSimultaneous antagonism of P2X7 and A2A significantly reduces tumor growth and cell dissemination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Photon emission on day 14 in mice treated with placebo (PBS), the P2X7 antagonist AZ10606120 (2mg/Kg), the A2A antagonist SCH58261 (1 mg/Kg), and both drugs. CT26 Luc2 cells were injected into the tail vein of mice (n=6 per condition) and luminescence emission is presented as total flux (p/s). (B) Images of CT26 Luc2 cells' luminescence emission were captured thanks to a luminometer for small animals on day 14. (C) Systemic levels of the pro-inflammatory cytokine IL-17 in mice injected intravenously with CT26 Luc2 and treated with placebo (PBS), the P2X7 antagonist AZ10606120 (2mg/Kg), the A2A antagonist SCH58261 (1 mg/Kg), and both drugs (n= 6 per condition). (D) Area of lung metastasis in mice injected intravenously with CT26 Luc2 and treated with the different drugs expressed as a percentage of total lung area (n= 6 per condition). Representative hematoxylin/eosin staining of lungs from mice treated with (E) placebo, (F) the P2X7 antagonist AZ10606120 (2mg/Kg), (G) the A2A antagonist SCH58261 (1 mg/Kg), and (H) combination of the two drugs. *p\u0026lt;0.05, **p\u0026lt;0.001, ***p\u0026lt; 0.0001, ****p\u0026lt; 0.00001. (I) \u003cem\u003eEx vivo\u003c/em\u003e tumor volume at day 14 of mice subcutaneously injected with CT26 (n=12 per condition) and treated with placebo (PBS), the P2X7 antagonist AZ10606120 (2mg/Kg), the A2A antagonist SCH58261 (1 mg/Kg) and both drugs. (J) Representative masses on day 14. (K) Systemic level of the pro-inflammatory cytokine IL-17 in mice subcutaneously injected with CT26 (n= 4 per condition).\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/4f01fd0a23009250e4edd756.png"},{"id":68203827,"identity":"cf2f3fa6-41f3-42e2-beb4-37a466ad3d08","added_by":"auto","created_at":"2024-11-04 16:01:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1894067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2X7 and A2A expression is decreased by double antagonism of the receptors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePercentage of lung cells positive for P2X7 (A) and A2A (F) staining in mice intravenously injected with the CT26 Luc 2. Representative immunohistochemistry images of mice treated with placebo (B, G), P2X7 antagonist AZ10606120 (C, H), A2A antagonist SCH58261 (D, I), or both drugs (E, J) (n= 4 per condition). *p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/aab8ed94c4a6a2392cac333f.png"},{"id":68203828,"identity":"a7359188-886c-4f6a-a155-1cde1fa36713","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":973729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2X7, CD39, CD73, and A2A expression significantly increased in the advanced stages of CRC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mRNA expression of P2X7A (A, B), P2X7B (C, D), CD39 (E, F), CD73 (G, H), and A2A (I, J) was evaluated in the cDNAs of 158 patients with CRC subdivided into stage I (n=24), stage II (n=50), stage III (n=52), and stage IV (n=32) which comprised 11 samples derived from metastases in organs other than the colon. (K) Spearman's correlation coefficient among P2X7A, P2X7B, CD39, CD73, and A2A was evaluated in CRC metastatic patients. (L) Spearman's correlation coefficient was evaluated between P2X7 and A2A expression in colon adenocarcinoma samples obtained from the Cancer Genome Atlas database. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/029ea88da6644dc10d0ab7b1.png"},{"id":68203834,"identity":"26b74185-dac8-4b10-9884-673927cebae5","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2867370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2X7 and A2A expression is enhanced in PIRC rats and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAPC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-mutated CRC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA expression of (A) P2X7A, (B) P2X7B, (C) A2A, (D) CD39, and (E) CD73 in CRC patients subdivided into \u003cem\u003eAPC\u003c/em\u003e WT and \u003cem\u003eAPC\u003c/em\u003emutated groups (n=6 per condition). *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. Percentage of cells positive for P2X7 (F) and A2A (G) in the colons of WT and PIRC rats and PIRC tumors (n=4 per condition). Representative images of immunohistochemical staining for P2X7 and A2A in the colon of WT 1-year rats (H, K) and in the normal colon (I, L) and the tumor mass (J, M) of 1-year PIRC rats.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/763fd8acf859345e6239a2a4.png"},{"id":88312827,"identity":"d3d8e6b4-a2cf-47df-b932-731993f07cd4","added_by":"auto","created_at":"2025-08-05 07:12:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18650751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/0a34fb1c-e77b-47a9-aa3c-710fe3abaf65.pdf"},{"id":68204922,"identity":"d5cc8c58-60a1-4215-9eb6-8dd56fbe7ced","added_by":"auto","created_at":"2024-11-04 16:09:10","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":73612,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/2cf0d328c675b66174add222.tif"},{"id":68203823,"identity":"d856320c-e5f9-4006-a89a-6c9b25f33cfc","added_by":"auto","created_at":"2024-11-04 16:01:10","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":565454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/c681004ee0fc17cfc28d2baf.tif"},{"id":68203836,"identity":"0118ec7f-279f-47d3-916d-5da37222c06d","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4198710,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/eceb44639aa5dce900a891cc.tif"},{"id":68203833,"identity":"adfbf7c6-7bcb-427b-ab34-f4592d41dcdb","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18318,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/8900ed4162b664d27fe09f4f.docx"},{"id":68204924,"identity":"065f1dd5-9421-496b-a660-8a37fcba5cb1","added_by":"auto","created_at":"2024-11-04 16:09:11","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5587400,"visible":true,"origin":"","legend":"movie 1","description":"","filename":"video1.avi","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/f206c79c0118f141a679454e.avi"},{"id":68204923,"identity":"cdbdc081-981d-4325-a01e-ff71e061b659","added_by":"auto","created_at":"2024-11-04 16:09:10","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4393006,"visible":true,"origin":"","legend":"\u003cp\u003emovie 2\u003c/p\u003e","description":"","filename":"video2.avi","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/ff767536c1dd32b71bc34da2.avi"},{"id":68203837,"identity":"33017e7f-e859-491a-8594-3bcbb4d49bb1","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5548712,"visible":true,"origin":"","legend":"\u003cp\u003emovie 3\u003c/p\u003e","description":"","filename":"video3.avi","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/6b3ad7b63d5efdd61b2ba479.avi"},{"id":68203829,"identity":"2902f4e1-5c51-46d6-a680-0c0dcf86064b","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":365438,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedWBsfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/e2033b32d314daa69c7b65ae.tif"},{"id":68203831,"identity":"337c47ca-2b08-4407-8662-ba7bcbc9cad7","added_by":"auto","created_at":"2024-11-04 16:01:11","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":71052,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedWBssfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5287461/v1/add1e50843f4c61167978ad7.tif"}],"financialInterests":"(Not answered)","formattedTitle":"P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: role of the A2A/CD39/CD73 axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tumor microenvironment (TME) is rich in extracellular ATP (eATP) and its hydrolytic derivative adenosine, which promotes pro and anti-tumor pathways acting on tumor cells and immune infiltrates (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The best-characterized receptor for eATP in cancer is P2X7, which is expressed by both immune and tumor cells, promoting cancer growth (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), neovascularization (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), dissemination (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), and release of its ligand ATP (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), as well as activating tumor-eradicating immune responses (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). ATP and adenosine levels are strictly intertwined as, in the extracellular milieu, adenosine is produced from ATP by phosphates group loss mediated by the ectonucleotidases CD39 and CD73 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Interestingly, these enzymes and the primary adenosine receptor expressed in cancer (A2A) are emerging therapeutic targets in clinical trials designed to relieve immunosuppression and circumvent resistance to tumor immunotherapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In addition, in the TME, A2A promotes VEGF secretion, tumor cell proliferation, and dissemination (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Recent literature strongly supports the existence of crosstalk between the purinergic and adenosinergic systems in promoting cancer. For example, tumors growing in P2X7 null mice overexpress CD73, CD39, and A2A in either immune-infiltrating or tumor cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Moreover, the mechanisms leading to the effectiveness of anti-CD39 antibodies as immune system reactivating drugs depend on P2X7 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), highlighting the close link between key members of the purinergic system in the TME. All these findings prompted researchers to designate P2X7, CD39, CD73, and A2A as purinergic checkpoints (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExtracellular vesicles, including exosomes and microvesicles, have been identified as cancer transformation and progression mediators, influencing metastatic dissemination (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Cancer cells released from the primary tumor can engraft only in a favorable microenvironment known as the metastatic niche. The release of vesicles is a mechanism by which cancer cells influence the composition of distant extracellular microenvironments, causing the so-called metastatic niche preconditioning effect: reprogramming the secondary organ sites to favor tumor cell engraftment and, therefore, metastasis formation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, although extracellular vesicle blockade holds promise as a therapeutic approach to prevent metastasis, no drugs able to interfere with this process are available on the market, probably because most of the targets identified so far are intracellular (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe P2X7 receptor was long known for its ability to trigger the release of extracellular vesicles containing IL-1β, IL-18, and other cytokines from immune and nervous system cells (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We have recently demonstrated that melanoma cells can also release exosomes and microvesicles upon P2X7 stimulation. These particles are characterized by miRNA content that is profoundly different from spontaneously released vesicles (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Interestingly, vesicles released following P2X7 activation also contain ATP (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), thus suggesting that they might shape purinergic signaling in the TME. Indeed, it has been shown that extracellular vesicles can carry CD39 and CD73 and degrade ATP into immunosuppressive adenosine, supporting cancer growth (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, an in-depth investigation was missing linking the P2X7 /CD39/CD73 /A2A axis with extracellular vesicle release and the ability of these particles to influence ATP and adenosine in the TME to promote metastasis. Our study investigates P2X7-dependent vesicle secretion as a possible cause of colorectal cancer (CRC) metastasis and its antagonism as a new therapeutic approach to avoid cancer dissemination.\u003c/p\u003e \u003cp\u003eCRC is one of the most common cancers and the second leading cause of cancer death worldwide. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The survival rates of patients with early-stage disease are increasing because of the efficacy of surgery, which is often combined with adjuvant therapy. However, late-stage metastatic CRC remains a clinical challenge (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A bad prognosis due to metastasis is frequently due to mutations in the adenomatous polyposis coli (\u003cem\u003eAPC\u003c/em\u003e) gene associated with familiar forms of CRC but also often found in sporadic carcinomas (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Although P2X7, CD39, CD73, and A2A have been separately associated with CRC development and progression (\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) a systematic analysis of their crosstalk in metastatic spreading was missing.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eP2X7 blockade reduces the release of extracellular vesicles from colon carcinoma cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe recently demonstrated the release of extracellular vesicles and exosomes following P2X7 stimulation in melanoma cells and how their miRNA content affects cancer cell migration (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, we investigated whether a similar mechanism can be activated by the P2X7 receptor in colon carcinoma cells. At this aim, we selected CT26 and HCT-116 colon carcinoma cell lines that both express the P2X7 receptor (supplementary Fig.\u0026nbsp;1). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that stimulation of P2X7 with its most potent agonist, BZ-ATP, causes the release of particles detectable by confocal microscopy in CT26 colon carcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, see supplementary video 1). This phenomenon can also be activated in HCT-116 cells (see supplementary video 2). Interestingly, treatment with the P2X7 negative allosteric modulator AZ10606120 reduced particle release, as shown by confocal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D, see supplementary video 3) and particle detection analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Blockade with another receptor antagonist, A740003, which has a distinct chemical structure from AZ10606120, caused a comparable reduction in particle release, thus confirming the efficacy of receptor antagonism in decreasing vesicular release from cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The mean diameter of the detected particles, measured using nanosight technology, was approximately 150\u0026ndash;200 nm and did not vary upon P2X7 stimulation or antagonism (data not shown). Western Blot analysis of the vesicular content showed that particles released following ATP stimulation lost GM130 while gaining Alix staining compared to spontaneously released particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting P2X7 activation-dependent changes in the vesicular nature and content. Interestingly, these particles also expressed P2X7, CD39, CD73, and A2A, indicating that they may regulate the purinergic/adenosinergic axis in the TME (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Indeed, particles not only carried ATP, as demonstrated by quinacrine staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D), but also caused an increase in pericellular ATP levels, which were more abundant when the vesicles were collected following P2X7 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). These measurements were performed using the pmeLUC probe, which allows the measurement of extracellular ATP on the plasma membrane of cancer cells expressing it (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). These data were confirmed by measuring free ATP in the supernatant using a classical luciferin/ luciferase assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Interestingly, adenosine levels in the cell supernatants were also increased by treatment with particles isolated following P2X7 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eadministration of P2X7-released vesicles enhances colon carcinoma cell dissemination.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand whether extracellular vesicles released upon P2X7 activation could play a role in colon carcinoma metastatic processes, we analyzed the effect of their administration on CT26 cell mobility with a scratch test assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These experiments showed that only particles released upon P2X7 stimulation (ATP-VS) and not those spontaneously released (S-VS) were able to increase colon carcinoma cell spreading \u003cem\u003ein vitro\u003c/em\u003e and that administration of P2X7 agonists together with its antagonist prevented this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Similar results have been obtained \u003cem\u003ein vivo\u003c/em\u003e in a metastatic model obtained by injecting CT26 cells in the caudal vein of BALB/c syngeneic mice. Pretreatment of CT26 cells with vesicles released upon P2X7 activation significantly increased cancer cell spreading in the lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C), systemic levels of the pro-inflammatory tumor-promoting cytokine IL-17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and metastasis formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-H). Interestingly, receptor antagonism with AZ10606120 abrogated the vesicle-dependent dissemination to the lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, E, I) and IL-17 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), strongly suggesting that P2X7 is a druggable target to prevent vesicle-dependent colon carcinoma metastatic progression. Interestingly, co-administration of microparticles released upon P2X7 stimulation not only increased cancer spreading (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, C, E-I) but also tissue expression of both P2X7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E) and A2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-J) and this phenomenon was reversible upon P2X7 blockade (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, E, F J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eP2X7 A2A combined antagonism reduces colon carcinoma growth, dissemination, and circulating IL-17 in a syngeneic mice model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the possible role of adenosine and the A2A receptor in P2X7-dependent metastatic dissemination, we tested the effects of P2X7 and A2A double blockade \u003cem\u003ein vivo.\u003c/em\u003e P2X7 and A2A antagonism alone or in combination proved effective in reducing the spread of CT26 cells in our metastatic model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, D-I). P2X7 antagonist AZ10606120 and A2A antagonist SCH58261 were administered alone or in combination every three days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar results were obtained when measuring the IL-17 circulating levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The significant reduction in metastatic spreading and engraftment observed with either P2X7 or A2A blockade suggests both receptors favoring CRC dissemination. Interestingly, when mimicking, with the same cells and mice strain, a primary CRC model obtained by subcutaneous injection, only co-administration of both drugs was effective in reducing cancer growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J) and the levels of IL-17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Moreover, only the combined antagonism of both receptors reduced the expression of P2X7 and A2A in the lungs of the metastasis-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, when we performed similar experiments with HCT-116 human CRC cells in \u003cem\u003enude\u003c/em\u003e mice, we did not measure any effect of P2X7 and A2A blockade alone or in combination, either in subcutaneous or intravenous models (see supplementary Fig.\u0026nbsp;2), suggesting strong involvement of the immune response in the mechanism of action of the drugs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP2X7, CD39, CD73, and A2A are upregulated in metastatic colon carcinoma patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo understand whether our findings on the role of the purinergic adenosinergic axis could also be translated to patients, we analyzed the expression of P2X7, CD39, CD73, and A2A in an array of 158 CRC specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our analysis showed that upregulation of both P2X7 human isoforms, P2X7A and P2X7B (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) are associated with stage IV CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), a subset of patients characterized by a bad prognosis and metastatic dissemination. Similar results were obtained for CD39 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), CD73 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), and A2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). In our tested samples, the mRNA that best increased according to the CRC stage was CD73 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Interestingly, when we further analyzed data from stage IV CRC samples, subdividing them into those obtained from primary tumors versus metastatic specimens, we found upregulation of all purinergic checkpoints in secondary metastatic forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, D, F, H, J). In these samples, we also reported a positive correlation between the expression levels of P2X7A and P2X7B (Spearman's coefficient 0,74), P2X7A and A2A (Spearman's coefficient 0,45), P2X7B and A2A (Spearman's coefficient 0,34) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). A moderate positive correlation between P2X7 and A2A was confirmed by the analysis of CRC samples from the Atlas database (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). However, it was impossible to distinguish between P2X7A and B isoforms in this case.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAPC\u003c/b\u003e \u003cb\u003emutation in patient specimens and the PIRC rat correlates with P2X7 and A2A overexpression\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAPC\u003c/em\u003e Oncogene mutational status is often associated with bad prognosis and metastatic dissemination in CRC patients. Therefore, to further corroborate our data, we also analyzed P2X7A, P2X7B, CD39, CD73, and A2A expression in mRNA extracted from human CRC samples coming from \u003cem\u003eAPC\u003c/em\u003e WT versus \u003cem\u003eAPC\u003c/em\u003e mutated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-M). Interestingly, P2X7A, P2X7B, and A2A mRNA were increased in \u003cem\u003eAPC\u003c/em\u003e mutated tumors. In the same samples, the levels of CD39 and CD73 also showed a tendency to be upregulated in \u003cem\u003eAPC\u003c/em\u003e-mutated patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-M). To substantiate the association between P2X7 and A2A to \u003cem\u003eAPC\u003c/em\u003e mutational status, we took advantage of a murine genetic model of intestinal tumorigenesis: PIRC rats carrying an \u003cem\u003eAPC\u003c/em\u003e mutation. These rats spontaneously develop tumors in the colon that faithfully reproduce familiar and sporadic CRC in humans (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Immunohistochemistry demonstrated an upregulation of P2X7 and A2A in both the colon mucosa and colon tumors of PIRC rats as compared to the colon mucosa of WT rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-H). Interestingly, this upregulation appeared to involve both cancer and immune cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, H). In contrast, CD39 and CD73 levels were not altered in PIRC rats (see Additional Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-H).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRC still accounts for an high number of deaths worldwide (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Despite recent advancements in survival expectations owing to emerging therapeutic options the metastatic forms of CRC remain a clinical challenge (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). These premises formed the basis of our study, which aimed to associate vesicular release triggered by eATP and the purinergic axis formed by P2X7/CD39/CD73 and A2A with CRC metastatic dissemination. Indeed, extracellular vesicles play important roles in preconditioning the metastatic niche, transforming it into a hospitable, tumor-friendly milieu that supports the engraftment and growth of cancer cells (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). eATP and its degradation product, adenosine, have been associated with CRC development, immune escape, and dissemination (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, an association between P2X7-dependent vesicular release and accumulation of ATP and adenosine in the TME leading to facilitation of metastasis was missing. Our study shows that P2X7 stimulation causes the release of extracellular vesicles from CRC cells (ATP-VS) that can promote metastasis formation \u003cem\u003ein vivo\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Although we did not characterize ATP-VS in detail, we know that their content differs from that of particles spontaneously released from the same cells (S-VS) for the absence of GM 130 and the presence of Alix, suggesting that they are not apoptotic bodies and that they might include exosomes (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). P2X7 antagonists can revert ATP-mediated vesicular release, as demonstrated by confocal and nanosight experiments performed with receptor-blocking drugs of different chemical natures. Administration of ATP-VS increases both eATP and adenosine levels in CRC cell cultures. Interestingly, eATP concentration rises both in the vicinity of the plasmalemma, as measured by luciferase expressed in the outer layer of the plasma membrane (pmeLUC) (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), and in the supernatant of the cells. The increase in adenosine following ATP-VS administration was delayed compared with that of eATP, suggesting a hydrolytic origin. Indeed, ATP-VS carry CD39 and CD73, implying that they can enhance the production and accumulation of adenosine in the TME, as previously described in other oncologic contexts (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). We recently demonstrated that the ATP-VS released by melanoma cells and the miRNAs they carry favor cancer cell migration \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), and, we confirmed that a similar mechanism is activated in CRC. Moreover, we extended these findings to a live model of metastatic carcinogenesis, showing increased dissemination and engraftment in the lungs of CRC cells pre-treated with ATP-VS and intravenously injected in mice. More importantly, P2X7 antagonism can eliminate the metastatic advantage conferred by vesicles. The finding that blockade of P2X7-dependent vesicular release can prevent CRC dissemination is of particular relevance given that although inhibition of extracellular vesicle secretion holds promise for preventing tumor progression (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), no drugs that target vesicular secretion have been approved for human use (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Our data show that vesicles released upon P2X7 activation were more effective than those spontaneously released by tumor cells, allowing CRC metastasis. This effect could be due to the fact that these particles carry prometastatic miRNAs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), ATP (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and mitochondria (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), which can activate the metabolic activity of cells (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) and shape the immune response (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Moreover, the high concentrations of eATP present in the TME are compatible with continuous P2X7 activation, leading to vesicle release and even increase following most traditional oncological interventions, including chemotherapy and radiotherapy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), suggesting that ATP-VS could favor the relapse of oncologic conditions following common treatments. Finally, several P2X7 antagonists have already been administered in patients with non-oncologic diseases, including Chron's, with little to no side effects (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Therefore, they can be quickly transferred to a clinical setting to prevent vesicle-mediated metastasis.\u003c/p\u003e \u003cp\u003eThe levels of eATP and adenosine in the TME are interdependent because adenosine is generated from ATP via CD39 and CD73, which are also known to favor cancer growth through immune suppression. Recent evidence shows that the lack or inhibition of P2X7 alters eATP levels and modulates CD39, CD73, and adenosine receptor A2A in both immune-infiltrating and cancer cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) strongly suggesting that manipulation of P2X7 receptor activity affects the entire purinergic/adenosinergic axis in the TME. Indeed, we demonstrated that vesicles released upon P2X7 activation enhance both extracellular ATP and adenosine levels and that they carry not only P2X7 but also CD39, CD73, and A2A, suggesting that the ATP-VS influences the adenosinergic axis. Additionally, ATP-VS pretreatment of disseminating tumor cells enhanced the expression of both P2X7 and A2A receptors in tumors engrafting to the lungs. To explore whether the mechanisms driving P2X7-dependent metastasis also influenced the pro-tumoral adenosinergic pathway, we investigated the effects of concurrent blockade of P2X7 and A2A receptors \u003cem\u003ein vivo\u003c/em\u003e. P2X7 and A2A antagonists, alone or in combination, were able to reduce the metastatic spread of CT26 CRC cells in a syngeneic mouse model. Thus confirming the importance of both receptors in the promotion of CRC metastasis. Notably, double antagonism, although not causing any evident extra reduction in metastatic spreading, was the only treatment able to decrease the expression of both P2X7 and A2A in the engrafted tumors. Thus suggesting a more efficacious action in the downmodulation of purinergic checkpoints and hinting at an addictive effect of the double antagonism on the growth of metastases following engraftment. The administration of P2X7 or A2A antagonists alone was not sufficient to mitigate subcutaneous primary tumor growth, which was nonetheless significantly reduced by combined anti-P2X7/A2A blockade. Interestingly, none of the antagonists alone or in combination was able to reduce CRC growth in the \u003cem\u003enude\u003c/em\u003e mouse model devoid of T cell-mediated responses, implicating a central role of the immune system in their mechanism of action. In fact, in the fully immune-competent syngeneic model, circulating levels of IL-17 were also reduced by P2X7 and A2A antagonism, following a pattern similar to that observed for tumor growth and metastatic dissemination. IL-17 is a cytokine implicated in autoimmune diseases and the maintenance of colon homeostasis (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), which promotes CRC development, progression, and metastasis and reduces the efficacy of immune therapy (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Both P2X7 and A2A have been previously associated with Th17 cells and IL-17 secretion in different pathological contexts (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). However, to our knowledge, this is the first demonstration that blocking P2X7 and A2A, alone or in combination, can significantly reduce the levels of IL-17 in CRC murine models. Interestingly, in the metastatic context, IL-17 levels can also be upregulated by treatment with vesicles derived from CRC cells stimulated with the P2X7 agonist ATP, suggesting that the cargo of these vesicles comprising ATP and miRNAs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) plays a central role in modulating Th17 pathways.\u003c/p\u003e \u003cp\u003eTo corroborate our data in a patient cohort we demonstrated that the mRNAs levels of the two main P2X7 human isoforms, P2X7A and P2X7B, were upregulated in patients with stage IV CRC. Moreover, they almost doubled when comparing expression in primary stage IV tumors versus metastatic forms. These data confirm the association of both isoforms with the metastatic transformation of other tumors, such as neuroblastoma (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), melanoma (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), osteosarcoma (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), and prostate cancer (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Similar results were obtained for CD39, CD73, and A2A expression with CD73, which was also upregulated in stage III CRC samples. When performing a correlation analysis in metastatic specimens, P2X7A and P2X7B were strongly associated; however, there was also a moderate correlation between both isoforms and A2A. This correlation was confirmed by analysis of the Atlas database, which, unfortunately, does not allow for the distinction between P2X7 isoforms. In subsequent experiments, we investigated the correlation between the upregulation of P2X7 and A2A receptors and \u003cem\u003eAPC\u003c/em\u003e oncogene mutational status. Our findings revealed that mRNA expression of both P2X7A and P2X7B isoforms, as well as A2A, was significantly elevated in \u003cem\u003eAPC\u003c/em\u003e-mutated colorectal cancer patient samples compared to wild-type controls. Similar trends were observed in tumors spontaneously developing in the colonic mucosa of \u003cem\u003eAPC\u003c/em\u003e-mutated PIRC rats, showing upregulation of both receptors in mucosal, cancerous and immune cells. In contrast, the expression levels of CD39 and CD73 remained relatively unchanged in both the PIRC rat model and \u003cem\u003eAPC\u003c/em\u003e-mutated patients. To our knowledge, this is the first demonstration of P2X7 upregulation in a tumor that spontaneously develops in an oncogene-mutated murine model and the first association in cancer patients between P2X7 isoforms and \u003cem\u003eAPC\u003c/em\u003e mutations. \u003cem\u003eAPC\u003c/em\u003e mutations were recently correlated with a poor response to immunotherapy in CRC (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e), and given the role of both receptors in the cancer immune response (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), it is tempting to speculate that the upregulation of P2X7 and A2A in these patients could be partly responsible for this phenomenon. Moreover, although the number of \u003cem\u003eAPC\u003c/em\u003e-mutated patients we tested was limited, we noticed that those were more frequently characterized by metastatic spread in organs other than the liver (supplementary Table\u0026nbsp;1) and, therefore, more challenging to treat, were also those overexpressing P2X7 and A2A suggesting that a P2X7-A2A targeting therapy might be indicated to prevent metastatic spreading in this patient population. In conclusion, this study elucidates a novel mechanism by which vesicular release modulates ATP and adenosine levels in cancer, thereby promoting metastatic dissemination. Our findings present an exciting therapeutic opportunity to target tumoral vesicular release, potentially enhancing immune responses against cancer. This innovative approach could significantly advance the field of theranostics by providing new strategies for combating metastasis and improving patient outcomes.\u003c/p\u003e"},{"header":"Materials/Subjects and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures and transfection\u003c/h2\u003e \u003cp\u003eCT26 murine and HCT116 human colon carcinoma cell lines (EP-CL-0071, EP-CL-0096, CliniScience, Amsterdam, Netherlands) were cultured in RPMI 1640 (Carlo Erba Reagents, Milan, Italy) and McCoy's 5A (Euroclone, Milan, Italy) medium, respectively. Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Euroclone). CT26 and HCT116 cells were stably transfected with intracellular Luc 2 and plasma membrane pmeLUC luciferase probes using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Stably transfected cells were maintained with hygromycin B (0.2 mg/mL, Roche, Basel, Switzerland ) or geneticin (0.4 mg/mL, Sigma-Aldrich, Darmstadt, Germany). All cells were routinely tested using the Mycoplasma kit detection from Applied Biological Material (Richmond, Canada).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConfocal microscopy\u003c/h3\u003e\n\u003cp\u003eCT26 and HCT116 cells were seeded onto 24 mm glass coverslips (Thermo Fisher Scientific), and loaded with 1\u0026micro;M plasma membrane dye PKH26GL (Sigma-Aldrich) or FM4-64 (Thermo Fischer Scientific) plus 1\u0026micro;M Quinacrine dihydrochloride (Q3251, Sigma Aldrich) for ATP and nucleic acid staining. Live single-cell imaging was performed with an Olympus FV3000 confocal microscope using the FV31S-SW software (Olympus, Tokyo, Japan). The excitation wavelengths were 445nm and 594 nm, respectively. Emission was measured in the 610\u0026ndash;710 nm range for red staining and in the 460\u0026ndash;500 nm range for green staining.\u003c/p\u003e\n\u003ch3\u003eVesicles concentration\u003c/h3\u003e\n\u003cp\u003eVesicles were concentrated from the cell culture supernatant by centrifugation. 10 x 10\u003csup\u003e5\u003c/sup\u003e cells were incubated in saline solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO\u003csub\u003e4,\u003c/sub\u003e 1 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 20 mM HEPES, 5.5 mM glucose, 5 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4) with or without 3mM ATP for 30 minutes at 37\u0026deg;C with 5% CO\u003csub\u003e2,\u003c/sub\u003e and in some conditions, pre-treated for 10 minutes with the P2X7 antagonists AZ10606120 (5 \u0026micro;M) (Tocris Bioscience, Bristol, UK) or A740003 (20 \u0026micro;M) (Tocris Bioscience). The supernatant was first depleted of cells and debris by centrifugation at 2000 x g for 10 minutes at 4\u0026deg;C. The vesicles were then pelleted at 20000 x g for one hour at 4\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eATP and adenosine measure\u003c/h2\u003e \u003cp\u003e2 x 10\u003csup\u003e4\u003c/sup\u003e CT26 pmeLUC cells were seeded in a 96-well plate and used as the ATP sensors. Spontaneously released (S-VS) or ATP stimulated (ATP-VS) vesicles were collected from CT26 cells as described above and resuspended in 10 \u0026micro;L of PBS at a concentration of 3 \u0026micro;g/\u0026micro;l. D- luciferin (Promega, Madison, Wisconsis, USA) was added to the wells containing CT26 pmeLUC at a concentration of 60 \u0026micro;g/mL. Basal luminescence emission was measured for 5 minutes using an IVIS Lumina Luminometer (Perkin Elmer, Waltham, Massachusetts, USA). Vesicles or 10 \u0026micro;L of PBS as a control were added to the cells, and luminescence emission was acquired for additional 5 minutes. Photon emission was quantified using the Living Image\u0026reg; Software (Perkin Elmer) as total photons/seconds (p/s). Changes in ATP concentration were expressed as a fold increase on basal luminescence emission. ATP and adenosine levels were measured in the supernatant of CT26 cells 30 and 90 minutes after the addition of vehicle (CTR), S-VS, or ATP-VS using ENLITEN rLuciferin/Luciferase reagent (Promega) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) or adenosine assay kit (Cell Biolabs, Inc. MET-5090, San Diego, California, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eS-VS and ATP-VS were collected in RIPA buffer plus Halt\u0026trade; Protease and Phosphatase inhibitor cocktail EDTA-free 100 x (Sigma-Aldrich). Protein lysates were loaded and separated on 4\u0026ndash;12% NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane, and incubated overnight with the following primary antibodies: anti-GM130 1:500 (Exosomal Marker Antibody Sample Kit, Cell Signaling Technology), anti-Alix 1:500 (Exosomal Marker Antibody Sample Kit, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-P2X7 1:300 (P8232, Sigma Aldrich) anti-CD73 1:250 (Bioss Antibodies, Woburn, Massachusetts, USA ), anti-CD39 1:500 (Cohesion Bioscience, London, UK) anti-A2A (SC32261, Santa Cruz Biotechnology, Dallas, Texas, USA). Incubation with secondary anti-rabbit (Thermo Fisher Scientific) or anti-mouse (Thermo Fisher Scientific) antibodies (1:2000) was performed for 1 hour. Protein bands were visualized using the ECL HRP Chemiluminescent Substrate ETA \u003cem\u003eC\u003c/em\u003e ULTRA 2.0 (Cyanagen Srl, Bologna, Italy) with a Licor C-Digit Model 3600. Expression of P2X7 and A2A in cell lysates from CT26-Luc2 and HCT116-Luc2 cells was demonstrated by immunoblot as previously described.(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eNanoparticle tracking analysis\u003c/h3\u003e\n\u003cp\u003eVesicles were resuspended in 60 \u0026micro;L of filtered PBS and diluted 1:100 or 1:500 in PBS. Vesicles were tracked using the Nanosight system (Nanosight LM10, Malvern, UK), according to the manufacturer's instructions. Data were analyzed using the NanoSight Software NTA 3.2 Dev Build 3.2.16.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScratch recovery assay\u003c/h2\u003e \u003cp\u003eA Wound Healing Assay (ab242285, Abcam, Cambridge, UK) was used for scratch tests in serum-free RPMI 1640 medium. CT26 cells were seeded in 24-well plates containing inserts. When they reached confluence, the insert was removed to generate a 0.9 mm wound field. Cells were treated with 10 \u0026micro;l of PBS (control), S-VS, ATP-VS, or ATP-VS collected following pretreatment with the P2X7 antagonist AZ10606120. Pictures of scratches were acquired at time 0 (T0) and after 24 and 48 hours (T24, T48) with a phase-contrast optical DM IL Led Leica Microsystem microscope (LEICA ICC50 HD, Wetzlar, Germany). ImageJ Software was used for scratch measurement. The percentage closure was calculated by comparison with T0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMurine models\u003c/h2\u003e \u003cp\u003eA syngeneic experimental metastasis model was established by injecting 2.5 x 10\u003csup\u003e5\u003c/sup\u003e CT26 Luc2 cells into the tail vein of BALB/c 4\u0026ndash;6 weeks-old female mice (Envigo, Udine, Italy). Mice were randomized into four groups of 6 animals each, and the operator was blinded to the allocation group. In a first set of experiments CT26 Luc2 cells were intravenously injected with vesicles collected as described in the \"Vesicles concentration\" paragraph. Cell dissemination was monitored every 72 hours by measuring luciferase Luc2 photon emission using an IVIS Lumina Luminometer (Perkin Elmer) as described previously (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In a second set of experiments, animals were treated every 72 hours with an i.p. injection of placebo (PBS\u0026thinsp;+\u0026thinsp;0.002% DMSO), the P2X7 antagonist AZ10606120 (2 mg/Kg) (Tocris Bioscience), the A2A antagonist SCH58261 (1 mg/Kg) (Tocris Bioscience), and a combination of both antagonists. The same antagonist administration schedule was also employed in a xenotransplant model obtained by tail vein injection of 2 x 10\u003csup\u003e6\u003c/sup\u003e HCT-116-Luc2 cells in \u003cem\u003enude/nude\u003c/em\u003e mice (Envigo). Mice were randomized into four groups of 7 animals each, and the operator was blinded to the allocation group. To mimic primary colon carcinoma, 5 x 10\u003csup\u003e5\u003c/sup\u003e CT26 cells were subcutaneously injected into 6-week-old BALB/c female mice (Envigo). The mice were randomized into four groups of 12 animals, and the operator was blinded to the allocation group. After five days, animals were treated every 72 hours with an intraperitoneal (i.p.) injection of placebo (PBS\u0026thinsp;+\u0026thinsp;0.002% DMSO), the P2X7 antagonist AZ10606120 (2 mg/Kg) (Tocris Bioscience), the A2A antagonist SCH58261 (1 mg/Kg) (Tocris Bioscience), and a combination of both antagonists. Tumor size was determined as described in (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The same antagonist administration schedule was also employed in a xenotransplant model obtained by subcutaneous injection of 2 x 10\u003csup\u003e6\u003c/sup\u003e HCT-116-Luc2 cells in \u003cem\u003enude/nude\u003c/em\u003e mice (Envigo). Mice were randomized into four groups of 6 animals each, and the operator was blinded to the allocation group. Blood samples were collected and prepared as previously described (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). All animal procedures were approved by the University of Ferrara Ethics Committee and the Italian Ministry of Health (Italian D. Lgs 26/204) and were in accordance with generally accepted guidelines for the welfare and use of animals in cancer research (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Tissue slides from the colons of WT and PIRC rats and related tumors were obtained from archived materials available at Caderni's laboratory (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCytokine quantification\u003c/h2\u003e \u003cp\u003ePlasma levels of interleukin 17 were measured using the Simple Plex\u0026trade; Cartridge Kit with Ella Automated Immunoassay System (Biotechne, Minneapolis, Minnesota, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eTissue slides from mouse lungs were processed as previously described (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Images were acquired using the NIS-Element Software with a Nikon Eclipse H550L microscope (Nikon Europe, Amstelveen, Netherlands). Metastasis formation in the lungs was analyzed by quantifying the area of the samples covered by clearly distinguishable tumor cells that were visible by hematoxylin/eosin staining. The total area of metastasis is expressed as a percentage of the entire lung area. The areas were quantified using ImageJ Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R\u0026amp;D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology). The protocol used for tissue staining has been described previously (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Images were acquired using a Nikon Eclipse H550L microscope and the NIS-Element Software (Nikon). The percentage of positive cells was quantified using QuPath open-source software. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative Real-Time PCR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSamples from human colon carcinoma patients were obtained from five commercial arrays (TissueScan\u0026trade; cDNA arrays HCRT301, HCRT302, HCRT303, HCRT304, HCRT305, OriGene, Rockville, Maryland, USA). Samples in which mRNA levels for housekeeping genes were not detectable were excluded from the analysis. A total of 158 patients were analyzed and subdivided according to the diagnostic phase into stage I (n\u0026thinsp;=\u0026thinsp;24), stage II (n\u0026thinsp;=\u0026thinsp;50), stage III (n\u0026thinsp;=\u0026thinsp;52) and stage IV (n\u0026thinsp;=\u0026thinsp;32). Stage IV patient data were further analyzed according to the origin of the cDNA from the primary (n\u0026thinsp;=\u0026thinsp;21) or metastatic (n\u0026thinsp;=\u0026thinsp;11) specimens. CRC samples cDNA were used as a template for quantitative Real-Time PCR (qRT-PCR) using TaqMan\u0026reg;MGB probes, FAM\u0026trade; dye-labeled (20X), and TaqMan Gene Expression Master Mix 2X (Applied Biosystems, Waltham, Massachusetts, USA). Taqman custom probes and primers for P2X7A and P2X7B were previously described by Pegoraro et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Taqman predesigned probes for the other genes were, respectively: Hs00969556_m1 ENTPD1 for CD39, Hs00159686_m1 NT5E for CD73, and Hs00169123_m1 ADORA2A for A2A (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1, Applied Biosystems) was used as a housekeeping gene. qRT-PCR was performed using an AB PRISM 7300 Step One Real-Time PCR system (Applied Biosystems) as previously described (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Formalin-fixed paraffin-embedded tumor samples (n\u0026thinsp;=\u0026thinsp;12) from patients with colon carcinoma were collected according to a protocol approved by the Istituto Romagnolo per lo studio dei tumori (IRST) Ethics Committee (CEROM IRST IRCCS-AVR, protocol code: IRST B125 approved on the 19th of March 2021). All patients signed an informed consent form before surgery. No specific inclusion or exclusion criteria were set. Patients' information is summarized in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Patients were stratified according to \u003cem\u003eAPC\u003c/em\u003e mutational status evaluated by whole exome sequencing (\u003cem\u003eAPC\u003c/em\u003e variants were considered with a variant allele frequency of 5% or greater). Total RNA was extracted from paraffin-embedded tissue sections using the Maxwell RSC RNA FFPE Kit (Promega, cat. no. AS1440) on a Maxwell CSC 48 automated nucleic acid extraction system (Promega) according to manufacturer instructions. Total RNA was quantified using the Qubit RNA High Sensitivity (HS) Assay Kit (Invitrogen, cat no. Q32852). 200 ng of total RNA were reverse-transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, cat. no Q32852). Real-time PCR was performed using the 7500 Real-time PCR System (Applied Biosystem, USA) as previously described (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene expression correlation\u003c/h2\u003e \u003cp\u003eSpearman's correlation analysis among the genes tested in our stage IV metastatic patients' commercial datasets was performed using GraphPad Prism Software (GraphPad, La Jolla, California, USA). Moreover, P2X7 and A2A gene expression was evaluated in colon adenocarcinoma samples from the Cancer Genome Atlas database (TGCA) using the GEPIA web server (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Spearman's correlation coefficient was applied for the analysis calculation with a non-log scale, whereas the data were visualized using the log-scale axis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Significance was calculated assuming equal standard deviation and variance, with a two-tailed Student's t-test or ordinary one-way ANOVA performed using GraphPad Prism Software (GraphPad). For each \u003cem\u003ein vivo\u003c/em\u003e experiment, the group size and statistical power were selected following computation \u003cem\u003ea priori\u003c/em\u003e, based on previous data (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), using an online sample size calculator (clincalc.com/stats/simplesize.aspx). Statistical significance was set at P-values lower than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Prof. Anna Lisa Giuliani, Dr. Simonetta Falzoni, Dr. Mario Tarantini and Prof. Massimo Bonora for fruitful discussion, Mrs. Marzia Scarletti and Dr. Federica Poletti for technical assistance.\u0026nbsp;This article is based upon work from PRESTO COST Action CA21130, supported by COST (European Cooperation in Science and Technology) www.cost.eu; www.p2xcost.eu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe late FDV was a member of the Scientific Advisory Board of Biosceptre Ltd. (UK), and a consultant at Breye Therapeutics (Denmark), and at Crosslink Therapeutics Inc (USA). All other authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEA and AP conceived and designed the study and wrote, reviewed, and revised the manuscript. AP, EDM, LR, MZ, SC, LA, MG, and EA performed the experiments. AP, EDM, LR, MZ, SC, GC, PU, and EA analyzed and interpreted the data. MZ, PU, AP, GG collected and processed patients' samples. FDV and LA participated in the\u0026nbsp;experimental design. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients' samples were collected in accordance with the IRST Ethics committee (CEROM IRST IRCCS-AVR, protocol code: IRST B125 approved on the 19th of March 2021). All the patients had signed informed consent prior to surgery. The animal study was reviewed and approved by Organismo Preposto al Benessere Animale (OPBA, organism for Animal Wellbeing), University of Ferrara and Italian Ministry of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Italian Association for Cancer Research (AIRC) Grants to EA\u0026nbsp;(IG22837), the PUR-THER, TRANSCAN3 Project to EA. Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 20225LKPYA to EA and LA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDi Virgilio F, Sarti AC, Falzoni S, De Marchi E, Adinolfi E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat Rev Cancer. 2018;18(10):601-18.\u003c/li\u003e\n\u003cli\u003eChiarella AM, Ryu YK, Manji GA, Rustgi AK. Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment. Trends Cancer. 2021;7(8):731-50.\u003c/li\u003e\n\u003cli\u003eBai X, Li Q, Peng X, Li X, Qiao C, Tang Y, et al. P2X7 receptor promotes migration and invasion of non-small cell lung cancer A549 cells through the PI3K/Akt pathways. Purinergic Signal. 2023.\u003c/li\u003e\n\u003cli\u003eAdinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, et al. Expression of P2X7 receptor increases in vivo tumor growth. Cancer Res. 2012;72(12):2957-69.\u003c/li\u003e\n\u003cli\u003eAmoroso F, Capece M, Rotondo A, Cangelosi D, Ferracin M, Franceschini A, et al. The P2X7 receptor is a key modulator of the PI3K/GSK3beta/VEGF signaling network: evidence in experimental neuroblastoma. Oncogene. 2015;34(41):5240-51.\u003c/li\u003e\n\u003cli\u003ePegoraro A, De Marchi E, Ferracin M, Orioli E, Zanoni M, Bassi C, et al. P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells. Cell Death Dis. 2021;12(12):1088.\u003c/li\u003e\n\u003cli\u003eDe Marchi E, Orioli E, Pegoraro A, Sangaletti S, Portararo P, Curti A, et al. The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment. Oncogene. 2019;38(19):3636-50.\u003c/li\u003e\n\u003cli\u003eAdinolfi E, De Marchi E, Orioli E, Pegoraro A, Di Virgilio F. Role of the P2X7 receptor in tumor-associated inflammation. Curr Opin Pharmacol. 2019;47:59-64.\u003c/li\u003e\n\u003cli\u003eKepp O, Bezu L, Yamazaki T, Di Virgilio F, Smyth MJ, Kroemer G, et al. ATP and cancer immunosurveillance. EMBO J. 2021;40(13):e108130.\u003c/li\u003e\n\u003cli\u003eYegutkin GG, Boison D. ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain. Pharmacol Rev. 2022;74(3):797-822.\u003c/li\u003e\n\u003cli\u003eZanoni M, Pegoraro A, Adinolfi E, De Marchi E. Emerging roles of purinergic signaling in anti-cancer therapy resistance. Front Cell Dev Biol. 2022;10:1006384.\u003c/li\u003e\n\u003cli\u003eMerighi S, Battistello E, Giacomelli L, Varani K, Vincenzi F, Borea PA, et al. Targeting A3 and A2A adenosine receptors in the fight against cancer. Expert Opin Ther Targets. 2019;23(8):669-78.\u003c/li\u003e\n\u003cli\u003ede Araujo JB, Kerkhoff VV, de Oliveira Maciel SFV, de Resende ESDT. Targeting the purinergic pathway in breast cancer and its therapeutic applications. Purinergic Signal. 2021;17(2):179-200.\u003c/li\u003e\n\u003cli\u003eDe Marchi E, Pegoraro A, Turiello R, Di Virgilio F, Morello S, Adinolfi E. A2A Receptor Contributes to Tumor Progression in P2X7 Null Mice. Front Cell Dev Biol. 2022;10:876510.\u003c/li\u003e\n\u003cli\u003eYan J, Li XY, Roman Aguilera A, Xiao C, Jacoberger-Foissac C, Nowlan B, et al. Control of Metastases via Myeloid CD39 and NK Cell Effector Function. Cancer Immunol Res. 2020;8(3):356-67.\u003c/li\u003e\n\u003cli\u003eCasey M, Segawa K, Law SC, Sabdia MB, Nowlan B, Salik B, et al. Inhibition of CD39 unleashes macrophage antibody-dependent cellular phagocytosis against B-cell lymphoma. Leukemia. 2023;37(2):379-87.\u003c/li\u003e\n\u003cli\u003eDemeules M, Scarpitta A, Hardet R, Gonde H, Abad C, Blandin M, et al. Evaluation of nanobody-based biologics targeting purinergic checkpoints in tumor models in vivo. Front Immunol. 2022;13:1012534.\u003c/li\u003e\n\u003cli\u003eSohal IS, Kasinski AL. Emerging diversity in extracellular vesicles and their roles in cancer. Front Oncol. 2023;13:1167717.\u003c/li\u003e\n\u003cli\u003eJeppesen DK, Zhang Q, Franklin JL, Coffey RJ. Extracellular vesicles and nanoparticles: emerging complexities. Trends Cell Biol. 2023;33(8):667-81.\u003c/li\u003e\n\u003cli\u003eBecker A, Thakur BK, Weiss JM, Kim HS, Peinado H, Lyden D. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell. 2016;30(6):836-48.\u003c/li\u003e\n\u003cli\u003eCatalano M, O\u0026apos;Driscoll L. Inhibiting extracellular vesicles formation and release: a review of EV inhibitors. J Extracell Vesicles. 2020;9(1):1703244.\u003c/li\u003e\n\u003cli\u003eLombardi M, Gabrielli M, Adinolfi E, Verderio C. Role of ATP in Extracellular Vesicle Biogenesis and Dynamics. Front Pharmacol. 2021;12:654023.\u003c/li\u003e\n\u003cli\u003eD\u0026apos;Arrigo G, Gabrielli M, Scaroni F, Swuec P, Amin L, Pegoraro A, et al. Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein. J Extracell Vesicles. 2021;10(9):e12114.\u003c/li\u003e\n\u003cli\u003eVultaggio-Poma V, Falzoni S, Chiozzi P, Sarti AC, Adinolfi E, Giuliani AL, et al. Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation. Theranostics. 2022;12(2):859-74.\u003c/li\u003e\n\u003cli\u003eCarotti V, Rigalli JP, van Asbeck-van der Wijst J, Hoenderop JGJ. Interplay between purinergic signalling and extracellular vesicles in health and disease. Biochem Pharmacol. 2022;203:115192.\u003c/li\u003e\n\u003cli\u003eSedlak JC, Yilmaz OH, Roper J. Metabolism and Colorectal Cancer. Annu Rev Pathol. 2023;18:467-92.\u003c/li\u003e\n\u003cli\u003eWaldum H, Fossmark R. Inflammation and Digestive Cancer. Int J Mol Sci. 2023;24(17).\u003c/li\u003e\n\u003cli\u003eShin AE, Giancotti FG, Rustgi AK. Metastatic colorectal cancer: mechanisms and emerging therapeutics. Trends Pharmacol Sci. 2023;44(4):222-36.\u003c/li\u003e\n\u003cli\u003eCancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330-7.\u003c/li\u003e\n\u003cli\u003eKunzli BM, Bernlochner MI, Rath S, Kaser S, Csizmadia E, Enjyoji K, et al. Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer. Purinergic Signal. 2011;7(2):231-41.\u003c/li\u003e\n\u003cli\u003eCalik I, Calik M, Turken G, Ozercan IH. A promising independent prognostic biomarker in colorectal cancer: P2X7 receptor. Int J Clin Exp Pathol. 2020;13(2):107-21.\u003c/li\u003e\n\u003cli\u003eFeng Y, Xu X, Zhang J, Sanderson C, Xia J, Bu Z, et al. CD39(+) tumor infiltrating T cells from colorectal cancers exhibit dysfunctional phenotype. Am J Cancer Res. 2024;14(2):585-600.\u003c/li\u003e\n\u003cli\u003eMessaoudi N, Cousineau I, Arslanian E, Henault D, Stephen D, Vandenbroucke-Menu F, et al. Prognostic value of CD73 expression in resected colorectal cancer liver metastasis. Oncoimmunology. 2020;9(1):1746138.\u003c/li\u003e\n\u003cli\u003eYe H, Zhao J, Xu X, Zhang D, Shen H, Wang S. Role of adenosine A2a receptor in cancers and autoimmune diseases. Immun Inflamm Dis. 2023;11(4):e826.\u003c/li\u003e\n\u003cli\u003eDe Marchi E, Orioli E, Pegoraro A, Adinolfi E, Di Virgilio F. Detection of Extracellular ATP in the Tumor Microenvironment, Using the pmeLUC Biosensor. Methods Mol Biol. 2020;2041:183-95.\u003c/li\u003e\n\u003cli\u003ePegoraro A, De Marchi E, Adinolfi E. P2X7 Variants in Oncogenesis. Cells. 2021;10(1).\u003c/li\u003e\n\u003cli\u003eAdinolfi E, De Marchi E, Grignolo M, Szymczak B, Pegoraro A. The P2X7 Receptor in Oncogenesis and Metastatic Dissemination: New Insights on Vesicular Release and Adenosinergic Crosstalk. Int J Mol Sci. 2023;24(18).\u003c/li\u003e\n\u003cli\u003eFemia AP, Soares PV, Luceri C, Lodovici M, Giannini A, Caderni G. Sulindac, 3,3\u0026apos;-diindolylmethane and curcumin reduce carcinogenesis in the Pirc rat, an Apc-driven model of colon carcinogenesis. BMC Cancer. 2015;15:611.\u003c/li\u003e\n\u003cli\u003eVitali F, Tortora K, Di Paola M, Bartolucci G, Menicatti M, De Filippo C, et al. Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers. Sci Rep. 2022;12(1):1432.\u003c/li\u003e\n\u003cli\u003eCervantes A, Adam R, Rosello S, Arnold D, Normanno N, Taieb J, et al. Metastatic colorectal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2023;34(1):10-32.\u003c/li\u003e\n\u003cli\u003eZeineddine FA, Zeineddine MA, Yousef A, Gu Y, Chowdhury S, Dasari A, et al. Survival improvement for patients with metastatic colorectal cancer over twenty years. NPJ Precis Oncol. 2023;7(1):16.\u003c/li\u003e\n\u003cli\u003eBekaii-Saab TS, Barzi A, Cusnir M. Improving survival in metastatic colorectal cancer through optimized patient selection. Clin Adv Hematol Oncol. 2024;22 Suppl 4(5):1-20.\u003c/li\u003e\n\u003cli\u003eUrabe F, Patil K, Ramm GA, Ochiya T, Soekmadji C. Extracellular vesicles in the development of organ-specific metastasis. J Extracell Vesicles. 2021;10(9):e12125.\u003c/li\u003e\n\u003cli\u003eD\u0026apos;Antongiovanni V, Fornai M, Pellegrini C, Benvenuti L, Blandizzi C, Antonioli L. The Adenosine System at the Crossroads of Intestinal Inflammation and Neoplasia. Int J Mol Sci. 2020;21(14).\u003c/li\u003e\n\u003cli\u003eWelsh JA, Goberdhan DCI, O\u0026apos;Driscoll L, Buzas EI, Blenkiron C, Bussolati B, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13(2):e12404.\u003c/li\u003e\n\u003cli\u003eMaacha S, Bhat AA, Jimenez L, Raza A, Haris M, Uddin S, et al. Extracellular vesicles-mediated intercellular communication: roles in the tumor microenvironment and anti-cancer drug resistance. Mol Cancer. 2019;18(1):55.\u003c/li\u003e\n\u003cli\u003eIrep N, Inci K, Tokgun PE, Tokgun O. Exosome inhibition improves response to first-line therapy in small cell lung cancer. J Cell Mol Med. 2024;28(4):e18138.\u003c/li\u003e\n\u003cli\u003eFalzoni S, Vultaggio-Poma V, Chiozzi P, Tarantini M, Adinolfi E, Boldrini P, et al. The P2X7 Receptor is a Master Regulator of Microparticle and Mitochondria Exchange in Mouse Microglia. Function (Oxf). 2024;5(4).\u003c/li\u003e\n\u003cli\u003ePizzirani C, Ferrari D, Chiozzi P, Adinolfi E, Sandona D, Savaglio E, et al. Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells. Blood. 2007;109(9):3856-64.\u003c/li\u003e\n\u003cli\u003eLongo Y, Mascaraque SM, Andreacchio G, Werner J, Katahira I, De Marchi E, et al. The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation. Front Immunol. 2024;15:1360140.\u003c/li\u003e\n\u003cli\u003eIqbal J, Bano S, Khan IA, Huang Q. A patent review of P2X7 receptor antagonists to treat inflammatory diseases (2018-present). Expert Opin Ther Pat. 2024;34(4):263-71.\u003c/li\u003e\n\u003cli\u003eHuangfu L, Li R, Huang Y, Wang S. The IL-17 family in diseases: from bench to bedside. Signal Transduct Target Ther. 2023;8(1):402.\u003c/li\u003e\n\u003cli\u003eRazi S, Baradaran Noveiry B, Keshavarz-Fathi M, Rezaei N. IL-17 and colorectal cancer: From carcinogenesis to treatment. Cytokine. 2019;116:7-12.\u003c/li\u003e\n\u003cli\u003eLiu C, Liu R, Wang B, Lian J, Yao Y, Sun H, et al. Blocking IL-17A enhances tumor response to anti-PD-1 immunotherapy in microsatellite stable colorectal cancer. J Immunother Cancer. 2021;9(1).\u003c/li\u003e\n\u003cli\u003eD\u0026apos;Addio F, Vergani A, Potena L, Maestroni A, Usuelli V, Ben Nasr M, et al. P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes. J Clin Invest. 2018;128(8):3490-503.\u003c/li\u003e\n\u003cli\u003eTokano M, Matsushita S, Takagi R, Yamamoto T, Kawano M. Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor. Brain Behav Immun Health. 2022;26:100544.\u003c/li\u003e\n\u003cli\u003eWang L, Wan H, Tang W, Ni Y, Hou X, Pan L, et al. Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17 cells in allergic asthma. Clin Respir J. 2018;12(1):149-57.\u003c/li\u003e\n\u003cli\u003eTattersall L, Shah KM, Lath DL, Singh A, Down JM, De Marchi E, et al. The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties. J Bone Oncol. 2021;31:100398.\u003c/li\u003e\n\u003cli\u003eSong H, Arredondo Carrera HM, Sprules A, Ji Y, Zhang T, He J, et al. C-terminal variants of the P2X7 receptor are associated with prostate cancer progression and bone metastasis - evidence from clinical and pre-clinical data. Cancer Commun (Lond). 2023;43(3):400-4.\u003c/li\u003e\n\u003cli\u003eLi B, Zhang G, Xu X. APC mutation correlated with poor response of immunotherapy in colon cancer. BMC Gastroenterol. 2023;23(1):95.\u003c/li\u003e\n\u003cli\u003eWorkman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. Guidelines for the welfare and use of animals in cancer research. Br J Cancer. 2010;102(11):1555-77.\u003c/li\u003e\n\u003cli\u003eBankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Sci Rep. 2017;7(1):16878.\u003c/li\u003e\n\u003cli\u003ePegoraro A, Orioli E, De Marchi E, Salvestrini V, Milani A, Di Virgilio F, et al. Differential sensitivity of acute myeloid leukemia cells to daunorubicin depends on P2X7A versus P2X7B receptor expression. Cell Death Dis. 2020;11(10):876.\u003c/li\u003e\n\u003cli\u003eZanoni M, Sarti AC, Zamagni A, Cortesi M, Pignatta S, Arienti C, et al. Irradiation causes senescence, ATP release, and P2X7 receptor isoform switch in glioblastoma. Cell Death Dis. 2022;13(1):80.\u003c/li\u003e\n\u003cli\u003eTang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45(W1):W98-W102.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5287461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5287461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular vesicle-driven cancer metastasis represents a therapeutic challenge due to the lack of effective blocking drugs. This study reveals a unique mechanism involving the P2X7 receptor and the A2A/CD39/CD73 axis, which affects ATP and adenosine levels in cancer via vesicular release, thereby enhancing metastasis. It also introduces a novel P2X7-based therapeutic approach to target tumor vesicular release. Indeed, activation of P2X7 on colon carcinoma cells induced the release of extracellular vesicles carrying P2X7, A2A, CD39, and CD73, resulting in significantly elevated ATP and adenosine levels within the tumor microenvironment. These vesicles enhanced colon carcinoma metastatic potential and systemic IL-17 production when administered \u003cem\u003ein vivo\u003c/em\u003e, effects that were successfully mitigated through P2X7 antagonism, which also reduced A2A levels in the metastatic niche. Treatment with P2X7 and A2A antagonists (AZ10606120 and SCH58261) markedly inhibited cancer growth and prevented tumor dissemination in an immune response-dependent manner. Finally, expression levels of P2X7, CD39, CD73, and A2A mRNAs were significantly higher in stage IV metastatic colon carcinoma patients. Furthermore, P2X7 and A2A expression increased in \u003cem\u003eAPC\u003c/em\u003e-mutated tumors and in spontaneous neoplasias within the colon mucosa of \u003cem\u003eAPC\u003c/em\u003e-mutated PIRC rats. Our study highlights the close interconnection between P2X7, A2A, CD39, and CD73 in colon carcinoma metastases. It identifies P2X7-dependent vesicle secretion as a new mechanism that favors metastatic dissemination and offers an innovative immunotherapeutic approach that targets vesicular release. Furthermore, we establish a first-time association between P2X7 and A2A overexpression and \u003cem\u003eAPC \u003c/em\u003eoncogene mutations, suggesting that these receptors could serve as potential biomarkers for advanced colon carcinoma.\u003c/p\u003e","manuscriptTitle":"P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: role of the A2A/CD39/CD73 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-04 16:01:05","doi":"10.21203/rs.3.rs-5287461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-01-03T14:22:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-12-30T10:32:02+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-13T13:10:31+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-08T10:29:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-04T08:16:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-10-28T12:30:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-21T10:59:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-18T07:49:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-10-18T07:49:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9db84d7b-f251-4d38-8004-223ab585524a","owner":[],"postedDate":"November 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39503632,"name":"Biological sciences/Cancer/Metastasis"},{"id":39503633,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":39503634,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":39503635,"name":"Biological sciences/Cell biology/Cell signalling/Ion channel signalling"},{"id":39503636,"name":"Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules"}],"tags":[],"updatedAt":"2025-08-05T07:12:23+00:00","versionOfRecord":{"articleIdentity":"rs-5287461","link":"https://doi.org/10.1038/s41419-025-07897-2","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-08-04 04:00:00","publishedOnDateReadable":"August 4th, 2025"},"versionCreatedAt":"2024-11-04 16:01:05","video":"","vorDoi":"10.1038/s41419-025-07897-2","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07897-2","workflowStages":[]},"version":"v1","identity":"rs-5287461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5287461","identity":"rs-5287461","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}