{"paper_id":"bcd7cda3-dacc-4b8c-bcf3-acd0e63e61dd","body_text":"Dual blockade of adenosine A2A and A 2B receptors is required to reverse NECA-induced immunosuppression in human macrophages: Implications for cancer immunotherapy | 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 Research Article Dual blockade of adenosine A2A and A 2B receptors is required to reverse NECA-induced immunosuppression in human macrophages: Implications for cancer immunotherapy Foteini Patera, Anna Malecka, Bryony Heath, Tajkia Musarrat, Aanchal Preet Kaur, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6975438/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Adenosine receptors are a target for cancer immunotherapy, with high levels of adenosine in the tumour microenvironment producing immunosuppressive effects. Most clinical trials in cancer are targeting the A 2A adenosine receptor, but evidence suggests that both the A 2A and A 2B receptors are important for determining the effect of adenosine in myeloid-lineage cells. We therefore studied the adenosine receptors involved in mediating the effect of the adenosine analogue 5'- N -Ethylcarboxamidoadenosine (NECA) on human monocyte-derived macrophages and assessed the down-stream effect of NECA-conditioned macrophages on T cell function. NECA-conditioning of human macrophages led to changes in cytokine and chemokine production resulting in a more ‘M2’ phenotype (decreased IL-12, IL-23, IL-6, TNFa and increased VEGF-A and IL-10). NECA-conditioned macrophages altered T cell phenotype in co-culture, impairing IFNγ production. Dual blockade of both A 2A and A 2B adenosine receptors was required to reverse the cytokine and chemokine changes seen in NECA-conditioned macrophages and to recover T cell IFNγ production. These data indicate that dual A 2A /A 2B receptor blockade will be required to re-polarise macrophages in a tumour environment to support cancer immunotherapeutic approaches. Macrophage adenosine receptor human cancer immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cancer immunotherapy has revolutionised management of some solid cancers, most notably metastatic melanoma. The most common immunotherapy in clinical practice is checkpoint blockade, involving monoclonal antibodies directed at the CTLA-4 and PD-1/PD-L1 receptors. Single (anti-PD1) or dual (anti-PD1 plus anti-CTLA4) immunotherapy for metastatic melanoma has increased median survival from 6 months in the chemotherapy era to over 5 years, with 52% 10 year melanoma-specific survival following dual immunotherapy[1]. These therapies enhance T cell activation, allowing immune attack of tumours. However, this only works in a limited proportion of cancers and patients. The tumour microenvironment (TME) itself is a key target to improve immunotherapy; altering this environment to support immune attack is a priority. Macrophages are an important mediator and determine whether the TME supports or suppresses immune attack of the tumour[2], with macrophages known to impact T cell function. Classically, macrophages have been considered as ‘M1’ (pro-inflammatory, anti-tumour, capable of producing high levels of pro-inflammatory IL-12) and ‘M2’ (anti-inflammatory, pro-angiogenic, tumour-supporting, capable of producing high levels of anti-inflammatory IL-10). In the TME, they are referred to as ‘tumour-associated macrophages’ (TAMs), are typically more ‘M2’-type and are detrimental to anti-cancer immune responses, producing anti-inflammatory, pro-tumoural factors such as IL-10 and VEGF. Repolarising these TAMs to a more immunogenic, anti-tumour phenotype would support immunotherapeutic approaches. Adenosine has been shown to be immunosuppressive, and can be present at higher levels in the TME than in physiologically normal tissues[3, 4]. Adenosine in the extracellular space is generated from ATP by the ectoenzymes CD39 and CD73, present on many cells in the TME including malignant cells, immune cells and stromal cells[5]. A non-canonical mechanism of adenosine generation also exists involving NAD+ which is converted to adenosine via CD38, CD203a and CD73[6]. Levels of CD73 (the terminal common enzyme for adenosine generation) are prognostic in a range of cancers[7]. Several studies have defined ‘adenosine high’ gene signatures and demonstrated either poor prognosis in tumours with this signature or responsiveness to adenosine-targeting therapies associated with this signature[8-10]. Pre-clinical models have also demonstrated that anti-PD1 therapy is less effective in CD73 hi tumours, and that anti-PD1 therapy induces upregulation of adenosine receptors[11]. Adenosine has a short half-life and is rapidly synthesised and degraded in tissues. For in vitro studies, where maintenance of steady levels is challenging due to rapid breakdown of exogenously-added adenosine, the more stable adenosine analogue 5'- N -Ethylcarboxamidoadenosine (NECA) is commonly used instead. Adenosine and NECA act via four G-protein coupled receptors: A 1 , A 2A , A 2B and A 3 , which are variably expressed on different cell types in the TME[12]. A 2A is a high-affinity receptor for adenosine, with an EC 50 of <1µM[13]. In contrast, A 2B has lower affinity with an EC 50 of 24µM[13]. Therefore, whilst the A 2B receptor may have a minimal role in healthy tissues with low levels of adenosine, it becomes significant in tumours with their higher levels of adenosine. It has been demonstrated that adenosine concentrations in the TME reach levels well above the EC 50 of the A2B receptor[4]. In T cells, the predominant receptor via which adenosine mediates immunomodulatory effects is A 2A [14-16]. Signalling via this receptor leads to suppression of effector T cell function and increased action of regulatory T cells[16] as well as impaired cytotoxic cytokine secretion[15]. Multiple adenosine-targeting agents are now in early-phase clinical trials for cancer, often as a combination approach[17]. Most of these target either the A 2A receptor or the CD73 ectoenzyme. Some studies have shown potentially encouraging results, including a small-molecule A 2A inhibitor +/-Atezolizumab in renal cancer, demonstrating safety and showing some partial responses in heavily pre-treated patients[9] and another study demonstrating benefit of adding anti-CD73 treatment to Durvalumab after concurrent chemo-radiotherapy for non-small cell lung cancer[18]. Others have started reporting with acceptable safety profiles and some indications of tumour response in both heavily pre-treated patients and in tumour types which are typically immunotherapy resistant[19, 20]. However, further publications show disappointing efficacy of A 2A or adenosine-generation targeting strategies[21-24]. Taken together, these early results indicate that it may be necessary to block multiple adenosine receptors to prevent adenosine-mediated immune suppression in the TME. In macrophages, data from murine systems shows that adenosine suppresses production of key immune-activating cytokines including IL-12 and TNFα but that the A 2A receptor alone does not mediate this effect[25]. Lack of production of these cytokines impedes anti-tumour immune attack by adaptive immune cells such as T cells. Gene expression data regarding adenosine receptors in myeloid cells is variable, but overall demonstrates that both the A 2A and A 2B receptors are expressed, [26-28]. Crucially, most studies have looked at A 2A or A 2B receptors in isolation, using either knockout models or single-receptor pharmacological inhibition, meaning that the relative contribution of A 2A and A 2B has not been explored. A number of studies have shown a switch towards an anti-inflammatory phenotype in human monocytes treated with adenosine (decrease in IL-12 and TNFα, increase in IL-10[29, 30]). In human monocyte-derived macrophages, it has been shown that adenosine increases VEGF production[31]. It has also been shown that in some tumour types, macrophages appear to mediate the effect of adenosine more potently than T cells[10]. We hypothesised that adenosine would polarise human macrophages towards an immunosuppressive phenotype via both the A 2A and A 2B receptors, and that dual receptor blockade would be required to reverse this. Here we present evidence that dual A 2A and A 2B receptor antagonism is required to reverse the effects of NECA on macrophages and prevent deleterious downstream effects on cells important for anti-tumour immunity. Materials and Methods Reagents: Endotoxin free reagents were used throughout. All media were based on RPMI 1640 with L-glutamine (Sigma). Macrophage medium contained v/v 10% FCS and 1% sodium pyruvate (Sigma), T cell medium v/v 10% FCS, 1% HEPES, 1% sodium pyruvate, 1% non-essential amino acids and 20µM 2-mercaptoethanol (all Sigma). Cytokines were recombinant human (rh) Granulocyte-macrophage colony-stimulating factor (GM-CSF, 20U/ml, Peprotech), rh Macrophage colony-stimulating factor (M-CSF, 10ng/ml, Immunotools), lipopolysaccharide (LPS, 1mg/ml, Sigma), interferon gamma (IFNγ, 1000U/ml, R&D). NECA (5′-(N-Ethylcarboxamido)- adenosine), SCH442416 and PSB603 were all from Tocris. Phytohaemaglutinin (PHA, 4μg/ml, Sigma). Macrophage generation: PBMC were separated from heparinised blood immediately after venepuncture by density centrifugation over Histopaque 1077 (Sigma). CD14+ cells were isolated using Miltenyi CD14+ microbead (purity >95% by flow cytometry). CD14+ monocytes were resuspended at 1x10 6 /ml and plated in ultra-low attachment plates (Corning costar) with either 5x10 6 /well (6 well plate) or 1x10 6 /well (24 well plate). For ‘M1’-type macrophages, GM-CSF was added at day 0 at 20U/ml and for ‘M2’-type macrophages, M-CSF was added at day 0 at 10ng/ml. Macrophages were fed at day 4 with equal volume medium + GM-CSF or M-CSF as appropriate. In some experiments, NECA, SCH442416 and/or PSB603 (all 1μM) were added to macrophage medium at d0 and d4 of differentiation. On day 7, macrophages were harvested by incubation on ice for 15 minutes in cold endotoxin-free PBS (Sigma), resuspended in macrophage medium (or T cell medium for co-culture experiments), plated and rested for 2 hours at 37 o /5%CO 2 prior to any drugs/stimulation. In vitro stimulation: Macrophages were resuspended at 5x10 5 /ml and plated at 5x10 4 per well in a standard 96-well plate. After resting for 2 hours macrophages were stimulated with LPS (1mg/ml) + IFNγ (1000U/ml) (GM-CSF macrophages) or LPS alone (M-CSF macrophages). In some experiments, NECA (1nM-10μM) and/or adenosine receptor inhibitor drugs (1μM) were added for 1 hour prior to stimulation. After 24hrs, supernatants were harvested for ELISA for IL-12p70 (BD Biosciences) and IL-10 (R&D) or for multiplex analysis using a custom-designed Luminex Human Magnetic Assay (R&D Systems). PBL:macrophage co-culture: Allogeneic peripheral blood leucocyte (PBL, CD14- fraction from monocyte isolations, frozen immediately after isolation at 2x10 7 /ml in 90% FCS + 10% DMSO) were defrosted and rested overnight at 2x10 6 /ml in T cell medium in 6 well plates (Costar). The next day, day 7 macrophages were harvested, plated at 1x10 3 , 3.3x10 3 , 1x10 4 or 2x10 4 /well in 100μl T cell medium in a 96-well plate and rested for 2 hours at 37 o /5%CO 2. PBL were then harvested, counted by haemocytometer, resuspended and added at 1x10 5 per well. PHA was added at 4μg/ml in a final volume of 200μl. On day 4, 50μl supernatant was removed for ELISA for IFNγ (BD Biosciences), IL-10 and IL-17 (both R&D). 50μl T cell medium containing 3 H-Thymidine was added immediately and cells cultured for a further 18hr then frozen at -20°C. Cells were subsequently transferred to 96-well unifilter microplates (PerkinElmer), dried overnight at room temperature then Microscint-O (PerkinElmer) added and read on a TopCount NXT (PerkinElmer). PBL without PHA stimulation were used as negative control and had undetectable levels of all cytokines and negligible thymidine incorporation (not shown). Statistics Data were analysed using Graphpad Prism with tests as indicated in figure legends. Summary data for multiple donors was normalised where appropriate to control condition for each donor then mean +/- SEM calculated for all donors together. In all figures, *=p<0.05, **=p<0.005 , ***=p<0.001. Results NECA modulates the function of both fully differentiated and differentiating human macrophages Our first aim was to confirm the effect of the adenosine analogue NECA on both fully differentiated and differentiating human macrophages. We used NECA throughout this study due to its longer half-life for in vitro experiments. To study the effect of NECA on fully differentiated macrophages, CD14+ monocytes were differentiated in medium containing either GM-CSF (to model an ‘M1’ phenotype) or M-CSF (to model an ‘M2’ phenotype). Macrophages were stimulated with LPS/IFNγ or LPS respectively to readout on their IL-12 and IL-10 cytokine production as a measure of their pro- or anti-inflammatory capacity. GM-CSF differentiated macrophages (GMCSF-Mφ) produced abundant IL-12 and smaller amounts of IL-10, whilst M-CSF differentiated macrophages (MCSF-Mφ) produced little/no IL-12 and higher levels of IL-10 (Figure 1a). Treatment with NECA for 1 hour prior to activation led to a significant concentration-dependent reduction in IL-12 production by GMCSF-Mφ and no significant changes in IL-10 (Figure 1bi, p<0.0001 and p=0.66 by one-way ANOVA respectively). For MCSF-Mφ, NECA surprisingly but reproducibly (and in contrast to murine data) produced a significant concentration-dependent decrease in IL-10 (Figure 1bii, p<0.0001 by one-way ANOVA). The A 2A receptor has been shown to be the most important adenosine receptor for mediating adenosine effects on T cells[16] and is the main current clinical target within the adenosine receptor family. In contrast, both A 2A and A 2B receptors are known to be expressed in murine and human myeloid cells[26-28]. We therefore used selective small molecule inhibitors of the A 2A and A 2B receptors (SCH442416 and PSB603 respectively, both 1μM) to investigate which adenosine receptor was mediating the effect of NECA in human macrophages. Treatment of macrophages with A 2A or A 2B antagonist alone did not result in any change in cytokine production (not shown). In GMCSF-Mφ, blockade of the A 2A or A 2B receptor alone prior to NECA treatment and activation produced no significant recovery in IL-12 production (Figure 1c left panel, top and middle). However, simultaneous blockade of both A 2A and A 2B receptors was required to significantly recover IL-12 secretion (Figure 1C left panel, bottom, p<0.001 at NECA concentrations of 100nM and above). Whilst the small increase in IL-10 secretion by NECA-treatment of GMCSF-Mφ did not reach statistical significance (Figure 1bi), there was a significant reduction in IL-10 following dual A 2A /A 2B blockade (Figure 1c middle panel, bottom). In MCSF-Mφ, the effect of NECA on IL-10 production was largely reversed by blockade of A 2B alone (Figure 1c right panel, middle, p<0.001 at NECA concentrations of 1μM and above) whilst A 2A blockade alone had very little effect (Figure 1c right panel, top). Thus, the ‘M2’ polarising effect of NECA on differentiated GMCSF-Mφ was only completely reversed by blockade of both A 2A and A 2B receptors, but blockade of A 2B alone largely reversed the effect of NECA on differentiated MCSF-Mφ. In the TME, monocytes exiting the vasculature and entering the tumour would be instantly exposed to higher levels of adenosine. Therefore, we studied the effect of NECA (1μM) on the differentiation of macrophages in order to more accurately mimic conditions experienced in the TME. Similar to the effects of NECA treatment after differentiation, the presence of NECA during differentiation significantly suppressed production of IL-12 from GMCSF-Mφ (Figure 2a). In contrast to NECA-treatment after differentiation, IL-10-secreting capacity was substantially increased when NECA was present during differentiation in both GMCSF-Mφ and MCSF-Mφ, in keeping with previous murine data (Figure 2a). This resulted in a significant reduction in the IL-12:IL-10 ratio in NECA-conditioned GMCSF-Mφ (Figure 2bi, p<0.05) and a mean 2.10-fold increase in IL-10 production (p<0.05, Figure 2bii). Dual blockade of A 2A and A 2B receptors is required to reverse the effect of adenosine on differentiating human macrophages Having established that both the A 2A and A 2B receptors mediate the effects of adenosine on fully differentiated human macrophages, we used the small molecule inhibitors during differentiation to establish which receptors were responsible for the repolarisation effect seen. There was no significant effect of adenosine receptor blockade alone on macrophage differentiation (not shown). Once again, combined A 2A and A 2B blockade was required to recover IL-12 production by NECA-conditioned GMCSF-Mφ (Figure 2ci, p<0.005). Notably, single A 2A or A 2B blockade did not recover IL-12 production in GMCSF-Mφ (Figure 2ci). In MCSF-Mφ, where NECA-conditioning significantly increased IL-10 production, combined A 2A and A 2B blockade significantly reduced IL-10 production (Figure 2cii). NECA has widespread effects on human macrophage function, mediated via both A 2A and A 2B receptors. Whilst IL-12 and IL-10 are an effective readout of ‘M1’ vs ‘M2’-type macrophages, many other cytokines and chemokines are produced which are important in a tumour context. We therefore wanted to establish what other effects NECA-conditioning had on macrophages and determine whether these were also mediated via both A 2A and A 2B receptors. We carried out a multiplex analysis of other cytokines and chemokines produced by macrophages stimulated after differentiation in the presence of NECA (1μM). Baseline comparison between control GM- Mφ and M- Mφ is shown in Supplementary Figure 1 and shows that, as expected, GM- Mφ produce more pro-inflammatory cytokines (IL-23, IL1β, IL-6 and TNFα) whilst M- Mφ produce more MMP7, MMP9 and VEGF-A. In M- Mφ, which are more representative of TAMs, we saw several marked and significant changes in cytokine/chemokine secretion after NECA-differentiation. IL-23, CCL-22, IL-6, TNFα , FGF-b, IL-1β and MMP2 were all significantly reduced in NECA-differentiated MCSF-Mφ (Figure 3). For the majority, significant recovery was only seen with dual A 2A and A 2B blockade (SCH442416 and PSB603, 1μM each). VEGF-A was markedly induced by NECA-differentiation in M- Mφ and again only fully reversed by dual A 2A /A 2B blockade. There was no significant effect on VEGF-C or VEGF-D (not shown). MMP2 production by NECA-differentiated M- Mφ was also significantly increased. Notably, GMCSF-Mφ were affected much less by NECA-polarisation than M- Mφ, with significant alterations only seen in Eotaxin 2 and CCL17 (Supplementary Figure 2). NECA-induced repolarisation of human macrophages impacts on downstream T cell function In order to determine whether NECA-polarisation of macrophages has a significant downstream impact on the effector immune cells which can attack and eliminate tumours, we carried out macrophage-PBL co-cultures. NECA was not included in the co-culture in order to assess the impact of the NECA-conditioned macrophages alone. IFNγ, IL-10 and IL-17 were measured to assess T cell polarisation. NECA-conditioned MCSF-Mφ significantly suppressed the ability of PBL to secrete IFNγ compared to control MCSF-Mφ (Figure 4ai (representative donor) and biii, p< 0.05). This was a dose-dependent effect (Figure 4b and supplementary figure 3) and was reversed by presence of A 2A /A 2B antagonism during macrophage differentiation (Figure 4ai and aiii, p=0.0410 and supplementary figure 3). IL-10 and IL-17 were not affected in MCSF-Mφ (Figure 4ai and iii). In keeping with the more modest effects seen on other cytokines and chemokines, NECA-conditioning of GMCSF-Mφ did not affect PBL IFNγ production (Figure 4ai and aii). Stimulation of A 2A receptors in T cells suppresses their ability to proliferate[32] so a potential mechanism for this was a reduced absolute number of PBL due to carry-over of NECA. However, we demonstrated that NECA-conditioned macrophages did not alter PBL proliferation compared to control macrophages (Figure 4ci & ii), confirming that the reduced IFNγ production was due to alterations in PBL phenotype in response to NECA-differentiated macrophages. Discussion Our data show that dual A 2A and A 2B receptor antagonism is required to completely reverse the effects of NECA on human macrophages. Furthermore, we demonstrate that the cytokine and chemokine alterations induced by NECA during macrophage differentiation have significant critical downstream effects on the adaptive immune cells which are required for tumour immunotherapy to be effective. The fact that these cytokine and chemokine changes were only reversed by dual A 2A and A 2B receptor blockade indicates that in tumours where there is a significant macrophage infiltration, A 2A blockade alone will not be sufficient to repolarise those macrophages to support tumour immunotherapy. In murine macrophages, it has been demonstrated using A 2A -receptor knockout studies that adenosine signalling via the A 2B receptor alone can suppress TNFα production[27]. However, in wild-type mice with expression of both A 2A and A 2B receptors, an A 2B receptor antagonist alone could not block the effects of adenosine, in keeping with our data. Another group also demonstrated in murine macrophage models that both A 2A and A 2B could mediate effects of adenosine when the other was not present[33]. In human lung macrophages (from patients undergoing lung resection for lung carcinoma, but taken from sections of lung distant to the tumour), a reduction in TNFα in response to NECA was seen[26]. Notably, whilst some reversal of this effect was seen with A 2A antagonism, it was not complete. A further paper on human monocytes also demonstrated partial recovery of TNFα production by human monocytes when NECA-treated in the presence of an A 2A antagonist alone [30]. However, crucially, none of these studies nor any others have examined the relative importance of A 2A and A 2B receptors when physiologically expressed and acting together in either murine or human macrophages. Our work is therefore the first to conclusively show that the effect of adenosine is mediated via both A 2A and A 2B receptors in human macrophages. It has been shown that when A 2A and A 2B receptors are co-expressed, the lower-affinity A 2B receptor tends to dictate the response to both NECA and adenosine[34, 35]. For example, in HEK 293G cells, the A 2A receptor contribution to cAMP responses to adenosine and NECA is very small, and the effect of A 2A receptor antagonism is limited[35]. Small signals can, however, be observed with selective A2A agonists[35]. We have generated both A 2A - and A 2B -specific fluorescent ligands[36, 37] and are characterising the relative levels of A 2A and A 2B in human macrophages generated under a variety of conditions. It is possible that different relative levels of A 2A and A 2B receptors in our GMCSF-Mφ and MCSF-Mφ would explain some of the differences in response to single antagonist treatment. Interestingly, in human lung macrophages it has been shown that A 2B expression is higher than A 2A in the resting state[26]. The majority of clinical trials in cancer targeting adenosine receptors are focussed on the A 2A receptor in combination with checkpoint inhibition. Further work is now needed to explore in relevant pre-clinical models the additional benefit of dual A2A/A 2B blockade in combination with checkpoint inhibition over checkpoint + A2A blockade alone. There is a dual A 2A /A 2B antagonist, AB928 for which phase 1b/2 studies are ongoing and showing encouraging efficacy[38]. Another compound targeting the A 2B receptor alone is currently in trials for non-small cell lung cancer after demonstration that A 2B blockade in combination with anti-PD1 was more beneficial than anti-PD1 alone[39]. A murine paper shows that combination of caffeine (a non-specific adenosine receptor antagonist) with anti-PD1 therapy leads to better tumour control in a carcinogen-induced tumour model and the B16F10 model than either therapy alone, with accompanying reduction in Treg infiltration and increase in both CD4 and CD8 cells in the tumour[40]. Another paper, whilst showing that caffeine was beneficial when combined with T-cell therapy, showed more substantial benefits with specific pharmacological inhibition of A 2A alone[41]. This suggests that whilst dual A 2A /A 2B blockade has promise, specific and high affinity pharmacological targeting will be required to realise the full potential of this approach. The latter paper was predominantly exploring A 2A receptor knockout/blockade in T cells but does also contain data supporting the concept that dual knockout of A 2A /A 2B even in T cells has an additional benefit over A 2A blockade alone[41]. In addition to the role of A 2B receptors on immune cells, there is renewed interest in this receptor more broadly within tumours as a potential therapeutic target[42]. Furthermore, in a study investigating T cell-adenosine interactions, whilst T-cell A 2A receptor expression was generally higher than A 2B , it is notable that in T cell populations that expressed higher than normal A 2B levels, A 2B inhibition could reverse adenosine-mediated suppression of effector function equally effectively as A2A inhibition[15]. Overall, our data and these other studies support the concept that dual A 2A /A 2B blockade may be required for cancer immunotherapy rather than targeting the A 2A receptor alone. Our multiplex analysis of secreted cytokines and chemokines shows that NECA has a much more marked effect on MCSF-Mφ than GMCSF-Mφ. GM-CSF is a strongly pro-inflammatory cytokine, and appears to ‘protect’ the macrophages from polarisation towards an M2 phenotype. Given that MCSF-Mφ are more representative of TAMs, the very marked effect of NECA seen on multiple secreted factors in this type of macrophage confirms the importance of developing methods to block the action of adenosine on macrophages in the TME. Whilst several of the cytokines and chemokines altered by NECA were T-cell polarising cytokines (IL-10, TNFα, IL-6 and IL-23), one of the most marked changes was in VEGF-A. It is unsurprising that NECA induces macrophages to produce more VEGF-A, since adenosine is upregulated in hypoxic environments and stimulation of angiogenesis is a mechanism to reverse hypoxia caused by inadequate blood supply. Previous work also noted an increase in VEGF production by human macrophages treated with adenosine[31]. They studied the impact of single adenosine receptor blockade and saw a significant reduction with A 2A blockade alone, however the combination of A 2A and A 2B blockade was not examined. Another study showed that in a murine model of oxygen-induced retinopathy, macrophage-specific Adora2a deletion did not reduce pathological neovascularisation[43]. One interpretation is that macrophages are not involved in this pathogenesis, but another is that adenosine also acts via A 2B in those macrophages and therefore deletion of only A 2A would not block its effects. In support of this hypothesis, there is evidence that the A 2B receptor is the predominant adenosine receptor controlling VEGF production[44]. CCL22, which was significantly reduced by NECA-conditioning, induces chemotaxis of T cells by binding to the CCR4 receptor. Decreased levels of CCL22 produced by TAMs exposed to high levels of adenosine would therefore impair migration of T cells into the tumour, which would be detrimental to current tumour immunotherapeutic effects. This multi-parameter effect of NECA on human macrophages underscores the importance of targeting adenosine receptors on TAMs to support immunotherapy. We saw a differential effect of NECA-conditioning during differentiation versus NECA treatment after differentiation of macrophages, suggesting that receptor expression and/or function varies in different types of myeloid cells. Our finding that IL-10 decreased with NECA-treatment in fully differentiated MCSF-Mφ was unexpected. Another paper has also noted a decrease in IL-10 when monocytes were treated with the adenosine analogue NECA immediately prior to stimulation[45]. As well as differential effects in different types of myeloid cells, it is likely that other cell types or culture conditions can modulate the effects of adenosine on human macrophages. For this reason, we are working to extend these findings into multi-cellular co-culture systems and explore the effects of other factors which are commonly encountered within the TME or during anti-cancer treatment, including hypoxia, extracellular matrix and radiotherapy. Hypoxia is particularly relevant in this context, given published evidence that A 2B receptors are preferentially upregulated in myeloid cells exposed to hypoxia[28]. In summary, this work is the first to clearly demonstrate that the A 2A and A 2B receptors are both non-redundant in mediating the effect of the adenosine analogue NECA on differentiating human macrophages, altering their functional phenotype in a way that modulates downstream immune responses. Dual targeting of A 2A and A 2B should therefore be explored further as a therapeutic strategy for tumour immunotherapy. Abbreviations ATP: adenosine triphosphate GM-CSF: Granulocyte-macrophage colony-stimulating factor GMCSF-Mφ: GM-CSF differentiated macrophages IFN: Interferon IL: Interleukin LPS: Lipopolysaccharide M-CSF: Macrophage colony-stimulating factor Mφ: macrophage MCSF-Mφ: M-CSF differentiated macrophages NECA: 5′-(N-Ethylcarboxamido)- adenosine PBL: Peripheral blood leucocytes PHA: Phytohaemaglutinin TAM: Tumour-associated macrophage TME: Tumour microenvironment TNF: Tumour-necrosis factor VEGF: Vascular endothelial growth factor Declarations Funding: This work was supported by a Cancer Research UK Postdoctoral Research Bursary for Clinicians (HF C50808/A24952), by the Medical Research Council (grant no. MR/W016176/1) and by the University of Nottingham. HF was an NIHR-funded Academic Clinical Lecturer in Medical Oncology. Competing interests: HF has received speaker’s honoraria from L’Oreal. None of the authors have a conflict of interest for this work. Author contributions: Conceived project: HF, SH; Designed experiments: HF, SH, AJ, PP, AM, TM; Carried out experiments: HF, FP, AM, BH, AK; Analysed data: HF, FP, BH, AK, TM; Wrote manuscript: HF; Reviewed manuscript: FP, AM, BH, TM, AK, PP, SH, AJ, HF Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate: All donors were recruited in accordance with Good Clinical Practice and the Declaration of Helsinki and provided written informed consent. Ethical approval was provided by University of Nottingham Medical School Ethics Committee (ref 161-1711). References Wolchok, J.D., et al., Final, 10-Year Outcomes with Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med, 2024. Caux, C., A Milestone Review on How Macrophages Affect Tumor Growth. Cancer Research, 2016. Blay, J., T.D. White, and D.W. 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Fong, L., et al., Adenosine 2A Receptor Blockade as an Immunotherapy for Treatment-Refractory Renal Cell Cancer. Cancer Discov, 2020. 10 (1): p. 40-53. Graziano, V., et al., Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma. J Immunother Cancer, 2023. 11 (8). Beavis, P.A., et al., CD73: A potential biomarker for anti-PD-1 therapy. Oncoimmunology, 2015. 4 (11): p. e1046675. Vijayan, D., et al., Targeting immunosuppressive adenosine in cancer. Nat Rev Cancer, 2017. Fredholm, B.B., et al., Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochem Pharmacol, 2001. 61 (4): p. 443-8. Alam, M.S., et al., A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence. Mucosal Immunol, 2009. 2 (3): p. 232-42. Mastelic-Gavillet, B., et al., Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8. J Immunother Cancer, 2019. 7 (1): p. 257. Ohta, A., A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment. Front Immunol, 2016. 7 : p. 109. Zahavi, D. and J. Hodge, Targeting Immunosuppressive Adenosine Signaling: A Review of Potential Immunotherapy Combination Strategies. International Journal of Molecular Sciences, 2023. 24 (10): p. 8871. Herbst, S.R., et al., COAST: An Open-Label, Phase II, Multidrug Platform Study of Durvalumab Alone or in Combination With Oleclumab or Monalizumab in Patients With Unresectable, Stage III Non–Small-Cell Lung Cancer. Journal of Clinical Oncology, 2022. 40 (29): p. 3383-3393. Bendell, J., et al., First-in-human study of oleclumab, a potent, selective anti-CD73 monoclonal antibody, alone or in combination with durvalumab in patients with advanced solid tumors. Cancer Immunology, Immunotherapy, 2023. 72 (7): p. 2443-2458. Lim, E.A., et al., Phase Ia/b, Open-Label, Multicenter Study of AZD4635 (an Adenosine A2A Receptor Antagonist) as Monotherapy or Combined with Durvalumab, in Patients with Solid Tumors. Clin Cancer Res, 2022. 28 (22): p. 4871-4884. Besse, B., et al., Biomarker-directed targeted therapy plus durvalumab in advanced non-small-cell lung cancer: a phase 2 umbrella trial. Nature Medicine, 2024. 30 (3): p. 716-729. Siddiqui, B.A., et al., Immune and pathologic responses in patients with localized prostate cancer who received daratumumab (anti-CD38) or edicotinib (CSF-1R inhibitor). Journal for ImmunoTherapy of Cancer, 2023. 11 (3): p. e006262. Falchook, G.S., et al., A phase 2 study of AZD4635 in combination with durvalumab or oleclumab in patients with metastatic castration-resistant prostate cancer. Cancer Immunol Immunother, 2024. 73 (4): p. 72. Coveler, L.A., et al., A Phase Ib/II Randomized Clinical Trial of Oleclumab with or without Durvalumab plus Chemotherapy in Patients with Metastatic Pancreatic Ductal Adenocarcinoma. Clinical Cancer Research, 2024. 30 (20): p. 4609-4617. Hasko, G., et al., Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms. FASEB J, 2000. 14 (13): p. 2065-74. Buenestado, A., et al., The role of adenosine receptors in regulating production of tumour necrosis factor-alpha and chemokines by human lung macrophages. Br J Pharmacol, 2010. 159 (6): p. 1304-11. Kreckler, L.M., et al., Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor. J Pharmacol Exp Ther, 2006. 317 (1): p. 172-80. Yang, M., et al., HIF-dependent induction of adenosine receptor A2b skews human dendritic cells to a Th2-stimulating phenotype under hypoxia. Immunol Cell Biol, 2010. 88 (2): p. 165-71. Link, A.A., et al., Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes. J Immunol, 2000. 164 (1): p. 436-42. Zhang, J.G., et al., The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes. Biochem Pharmacol, 2005. 69 (6): p. 883-9. Ernens, I., et al., Adenosine up-regulates vascular endothelial growth factor in human macrophages. Biochem Biophys Res Commun, 2010. 392 (3): p. 351-6. Sevigny, C.P., et al., Activation of adenosine 2A receptors attenuates allograft rejection and alloantigen recognition. J Immunol, 2007. 178 (7): p. 4240-9. Csoka, B., et al., Adenosine promotes alternative macrophage activation via A2A and A2B receptors. FASEB J, 2012. 26 (1): p. 376-86. Hinz, S., et al., Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors. Oncotarget, 2018. 9 (17): p. 13593-13611. Goulding, J., L.T. May, and S.J. Hill, Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603. Biochem Pharmacol, 2018. 147 : p. 55-66. Patera, F., et al., A novel and selective fluorescent ligand for the study of adenosine A<sub>2B</sub> receptors. Pharmacology Research & Perspectives, 2024. 12 (4). Comeo, E., et al., Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A(2A) Receptor. J Med Chem, 2020. 63 (5): p. 2656-2672. Wainberg, A.Z., et al., ARC-9: A randomized study to evaluate etrumadenant based treatment combinations in previously treated metastatic colorectal cancer (mCRC). Journal of Clinical Oncology, 2024. 42 (16_suppl): p. 3508-3508. Evans, J.V., et al., Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression. JNCI: Journal of the National Cancer Institute, 2023. 115 (11): p. 1404-1419. Tej, G., K. Neogi, and P.K. Nayak, Caffeine-enhanced anti-tumor activity of anti-PD1 monoclonal antibody. Int Immunopharmacol, 2019. 77 : p. 106002. Kjaergaard, J., et al., A2A Adenosine Receptor Gene Deletion or Synthetic A2A Antagonist Liberate Tumor-Reactive CD8(+) T Cells from Tumor-Induced Immunosuppression. J Immunol, 2018. Strickland, L.N., et al., The resurgence of the Adora2b receptor as an immunotherapeutic target in pancreatic cancer. Front Immunol, 2023. 14 : p. 1163585. Liu, Z., et al., Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis. Nat Commun, 2017. 8 (1): p. 584. Ryzhov, S., et al., Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production. Mol Pharmacol, 2014. 85 (1): p. 62-73. Le Moine, O., et al., Adenosine enhances IL-10 secretion by human monocytes. J Immunol, 1996. 156 (11): p. 4408-14. Additional Declarations No competing interests reported. Supplementary Files SupplementaryfiguresforCII.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6975438\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":508601633,\"identity\":\"126cbad6-6c7b-4dbf-9570-25b8543385ff\",\"order_by\":0,\"name\":\"Foteini Patera\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Nottingham\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Foteini\",\"middleName\":\"\",\"lastName\":\"Patera\",\"suffix\":\"\"},{\"id\":508601634,\"identity\":\"6d92418d-dd3f-4223-b548-f56f7e8db0a1\",\"order_by\":1,\"name\":\"Anna 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Nottingham\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Hester\",\"middleName\":\"A\",\"lastName\":\"Franks\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-06-25 13:53:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6975438/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6975438/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":90924473,\"identity\":\"f7ab02b8-4626-47db-a851-89105eb4ba4c\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:22:24\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":179841,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNECA modulates cytokine production by fully differentiated human macrophages via both A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2A\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e and A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2B\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e receptors\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ea) Macrophages differentiated in GM-CSF (GMCSF-Mφ) or M-CSF-containing medium (MCSF-Mφ) were stimulated with LPS or LPS/IFNγ respectively and secretion of IL-12 (black bar) and IL-10 (white bar) measured by ELISA in culture supernatants after 24hrs. (representative donor of n=25, mean +/- SEM of triplicate stimulation).\\u003c/p\\u003e\\n\\u003cp\\u003eb) \\u0026nbsp;GM-CSF (i) and M-CSF (ii) macrophages were treated with increasing concentrations of NECA prior to stimulation and cytokine analysis as in a) (summary of 9 donors each normalised to no NECA, mean +/- SEM, one-way ANOVA with post-hoc Dunnett’s multiple comparison compared to vehicle controls in the absence of NECA).\\u003c/p\\u003e\\n\\u003cp\\u003ec) GM-CSF and M-CSF macrophages were treated with an A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor antagonist (SCH442416, 1μM), an A\\u003csub\\u003e2B \\u003c/sub\\u003ereceptor antagonist (PSB603, 1μM) or both prior to addition of NECA, stimulation and cytokine analysis as in a) (summary of 5 donors each normalised to no NECA, mean +/- SEM, mixed-effects analysis with Sidak’s multiple comparisons test).\\u003c/p\\u003e\\n\\u003cp\\u003e*=p\\u0026lt;0.05, **=p\\u0026lt;0.005\\u0026nbsp;, ***=p\\u0026lt;0.001\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/14e941127976a3f3942e730a.png\"},{\"id\":90924472,\"identity\":\"2d36309c-69bc-406e-8d10-a10bfca034d0\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:22:24\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":112647,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNECA-conditioning of differentiating human macrophages polarises towards ‘M2’ type via both A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2A\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e and A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2B\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e receptors\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ea) Macrophages differentiated in GM-CSF (GMCSF-Mφ) or M-CSF-containing medium (MCSF-Mφ) +/- NECA (1μM) were stimulated with LPS or LPS/IFNγ respectively and secretion of IL-12 and IL-10 measured by ELISA in culture supernatants after 24hrs (representative donor of n=13, mean +/- SEM of triplicate stimulation, 2-tailed paired students T test).\\u003c/p\\u003e\\n\\u003cp\\u003eb) i) IL-12:IL-10 ratio for GMCSF-Mφ and ii) fold-change IL-10 for MCSF-Mφ both differentiated with/without NECA, stimulated and analysed as in a) (both n=7, 2-tailed paired students T test)\\u003c/p\\u003e\\n\\u003cp\\u003ec) Macrophages differentiated, stimulated and analysed as in a) +/- A\\u003csub\\u003e2A\\u003c/sub\\u003e inhibitor (A2Ai, SCH442416 1μM), A\\u003csub\\u003e2B\\u003c/sub\\u003e inhibitor (A2Bi, PSB603 1μM) or both during differentiation. Summary of 6 donors (GMCSF-Mφ) and 7 donors (MCSF-Mφ) normalised to no-NECA control (mean +/- SEM, Friedman Test with Dunn’s post test). Each symbol represents an individual donor.\\u003c/p\\u003e\\n\\u003cp\\u003e*=p\\u0026lt;0.05, **=p\\u0026lt;0.005\\u0026nbsp;, ***=p\\u0026lt;0.001\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/2df9d72008d0f439cf606622.png\"},{\"id\":90924475,\"identity\":\"faa39b31-3d35-4238-b45d-29b18fc8a2a4\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:22:24\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":142330,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNECA-conditioning of human M-CSF macrophages alters multiple cytokines and chemokines\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eM-CSF macrophages conditioned with NECA (1μM) +/- A\\u003csub\\u003e2A\\u003c/sub\\u003e inhibitor (A2Ai, SCH442416 1μM) and/or A\\u003csub\\u003e2B\\u003c/sub\\u003e inhibitor (A2Bi, PSB603 1μM) were stimulated with LPS for 24hrs and supernatants collected for Luminex analysis. Shown are single determinations of secretion for 8 independent donors (each symbol=one donor) with line at median. Statistical analysis using Friedman test with Dunn’s post –test, *=p\\u0026lt;0.05, **=p\\u0026lt;0.005\\u0026nbsp;, ***=p\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/1ef586fdd7ad2ba7571618f5.png\"},{\"id\":90924474,\"identity\":\"ee30ece1-37ed-4487-85f3-a0c885a6f8e8\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:22:24\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":148326,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNECA acts via A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2A\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e and A\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003e2B\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e receptors during differentiation to polarise human macrophages to impair T cell IFNγ responses\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMacrophages were differentiated in NECA-containing medium (1μM) +/- A2A inhibitor (A2Ai, SCH442416 1μM) and A2B inhibitor (A2Bi, PSB603 1μM) with either GM-CSF (GMCSF-Mφ) or M-CSF (MCSF-Mφ), harvested then co-cultured for 5 days with peripheral blood lymphocytes (PBL) along with PHA stimulation at 100:20, 100:10, 100:3 and 100:1 PBL:macrophage ratios. At 4 days, 50μl supernatant was collected for cytokine analysis via ELISA for IFNγ, IL-10 and IL-17, and replaced with 50μl medium containing thymidine for final 18hrs for proliferation analysis. a) IFNγ (top), IL-10 (middle) and IL-17 (bottom) secretion of i) mean +/- SEM of triplicate co-culture from 1 representative donor of 5 for GMCSF-Mφ and MCSF-Mφ, ii) \\u0026amp; iii) summary of each donor normalised to PBL + PHA alone GM-CSF and MCSF respectively, shown is mean for each donor (each symbol=one donor) with line at mean (One-way ANOVA with Dunnett’s multiple comparison test). b) IFNγ production by PBL co-cultured with NECA-conditioned MCSF-Mφ at different PBL:macrophage ratios (mean +/- SEM of data normalised as in Aiii), c) Proliferation analysis normalised to PBL + PHA alone for each donor (mean +/- SEM) for GMCSF-Mφ (i) and MCSF-Mφ (ii) (n=5 donors), *=p\\u0026lt;0.05, **=p\\u0026lt;0.005\\u0026nbsp;, ***=p\\u0026lt;0.001\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/be1dfc53deb49e34f0703fbc.png\"},{\"id\":90925667,\"identity\":\"aa9e8d5c-7ea3-451b-9152-9aa41998febe\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:30:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1429778,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/5f4408d3-0d91-437f-af6e-43282d557e4b.pdf\"},{\"id\":90924476,\"identity\":\"f19dc9c6-f89f-4db9-88e2-a74f4b85ea23\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 15:22:24\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":181968,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryfiguresforCII.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6975438/v1/b96de31681e8e1a917aa96d7.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Dual blockade of adenosine A2A and A 2B receptors is required to reverse NECA-induced immunosuppression in human macrophages: Implications for cancer immunotherapy\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCancer immunotherapy has revolutionised management of some solid cancers, most notably metastatic melanoma. The most common immunotherapy in clinical practice is checkpoint blockade, involving monoclonal antibodies directed at the CTLA-4 and PD-1/PD-L1 receptors. Single (anti-PD1) or dual (anti-PD1 plus anti-CTLA4) immunotherapy for metastatic melanoma has increased median survival from 6 months in the chemotherapy era to over 5 years, with 52% 10 year melanoma-specific survival following dual immunotherapy[1]. These therapies enhance T cell activation, allowing immune attack of tumours. However, this only works in a limited proportion of cancers and patients. The tumour microenvironment (TME) itself is a key target to improve immunotherapy; altering this environment to support immune attack is a priority. Macrophages are an important mediator and determine whether the TME supports or suppresses immune attack of the tumour[2], with macrophages known to impact T cell function. Classically, macrophages have been considered as ‘M1’ (pro-inflammatory, anti-tumour, capable of producing high levels of pro-inflammatory IL-12) and ‘M2’ (anti-inflammatory, pro-angiogenic, tumour-supporting, capable of producing high levels of anti-inflammatory IL-10). In the TME, they are referred to as ‘tumour-associated macrophages’ (TAMs), are typically more ‘M2’-type and are detrimental to anti-cancer immune responses, producing anti-inflammatory, pro-tumoural factors such as IL-10 and VEGF. Repolarising these TAMs to a more immunogenic, anti-tumour phenotype would support immunotherapeutic approaches.\\u003c/p\\u003e\\n\\u003cp\\u003eAdenosine has been shown to be immunosuppressive, and can be present at higher levels in the TME than in physiologically normal tissues[3, 4]. Adenosine in the extracellular space is generated from ATP by the ectoenzymes CD39 and CD73, present on many cells in the TME including malignant cells, immune cells and stromal cells[5]. A non-canonical mechanism of adenosine generation also exists involving NAD+ which is converted to adenosine via CD38, CD203a and CD73[6]. Levels of CD73 (the terminal common enzyme for adenosine generation) are prognostic in a range of cancers[7]. Several studies have defined ‘adenosine high’ gene signatures and demonstrated either poor prognosis in tumours with this signature or responsiveness to adenosine-targeting therapies associated with this signature[8-10]. Pre-clinical models have also demonstrated that anti-PD1 therapy is less effective in CD73\\u003csup\\u003ehi\\u003c/sup\\u003e tumours, and that anti-PD1 therapy induces upregulation of adenosine receptors[11]. \\u003c/p\\u003e\\n\\u003cp\\u003eAdenosine has a short half-life and is rapidly synthesised and degraded in tissues. For \\u003cem\\u003ein vitro \\u003c/em\\u003estudies, where maintenance of steady levels is challenging due to rapid breakdown of exogenously-added adenosine, the more stable adenosine analogue 5'-\\u003cem\\u003eN\\u003c/em\\u003e-Ethylcarboxamidoadenosine (NECA) is commonly used instead. Adenosine and NECA act via four G-protein coupled receptors: A\\u003csub\\u003e1\\u003c/sub\\u003e, A\\u003csub\\u003e2A\\u003c/sub\\u003e, A\\u003csub\\u003e2B\\u003c/sub\\u003e and A\\u003csub\\u003e3\\u003c/sub\\u003e, which are variably expressed on different cell types in the TME[12]. A\\u003csub\\u003e2A\\u003c/sub\\u003e is a high-affinity receptor for adenosine, with an EC\\u003csub\\u003e50\\u003c/sub\\u003e of \\u0026lt;1µM[13]. In contrast, A\\u003csub\\u003e2B\\u003c/sub\\u003e has lower affinity with an EC\\u003csub\\u003e50\\u003c/sub\\u003e of 24µM[13]. Therefore, whilst the A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor may have a minimal role in healthy tissues with low levels of adenosine, it becomes significant in tumours with their higher levels of adenosine. It has been demonstrated that adenosine concentrations in the TME reach levels well above the EC\\u003csub\\u003e50\\u003c/sub\\u003e of the A2B receptor[4]. In T cells, the predominant receptor via which adenosine mediates immunomodulatory effects is A\\u003csub\\u003e2A\\u003c/sub\\u003e[14-16]. Signalling via this receptor leads to suppression of effector T cell function and increased action of regulatory T cells[16] as well as impaired cytotoxic cytokine secretion[15]. \\u003c/p\\u003e\\n\\u003cp\\u003eMultiple adenosine-targeting agents are now in early-phase clinical trials for cancer, often as a combination approach[17]. Most of these target either the A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor or the CD73 ectoenzyme. Some studies have shown potentially encouraging results, including a small-molecule A\\u003csub\\u003e2A\\u003c/sub\\u003e inhibitor +/-Atezolizumab in renal cancer, demonstrating safety and showing some partial responses in heavily pre-treated patients[9] and another study demonstrating benefit of adding anti-CD73 treatment to Durvalumab after concurrent chemo-radiotherapy for non-small cell lung cancer[18]. Others have started reporting with acceptable safety profiles and some indications of tumour response in both heavily pre-treated patients and in tumour types which are typically immunotherapy resistant[19, 20]. However, further publications show disappointing efficacy of A\\u003csub\\u003e2A\\u003c/sub\\u003e or adenosine-generation targeting strategies[21-24]. Taken together, these early results indicate that it may be necessary to block multiple adenosine receptors to prevent adenosine-mediated immune suppression in the TME. \\u003c/p\\u003e\\n\\u003cp\\u003eIn macrophages, data from murine systems shows that adenosine suppresses production of key immune-activating cytokines including IL-12 and TNFα but that the A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor alone does not mediate this effect[25]. Lack of production of these cytokines impedes anti-tumour immune attack by adaptive immune cells such as T cells. Gene expression data regarding adenosine receptors in myeloid cells is variable, but overall demonstrates that both the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B \\u003c/sub\\u003ereceptors are expressed, [26-28]. Crucially, most studies have looked at A\\u003csub\\u003e2A \\u003c/sub\\u003eor A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors in isolation, using either knockout models or single-receptor pharmacological inhibition, meaning that the relative contribution of A\\u003csub\\u003e2A \\u003c/sub\\u003eand A\\u003csub\\u003e2B\\u003c/sub\\u003e has not been explored. A number of studies have shown a switch towards an anti-inflammatory phenotype in human monocytes treated with adenosine (decrease in IL-12 and TNFα, increase in IL-10[29, 30]). In human monocyte-derived macrophages, it has been shown that adenosine increases VEGF production[31]. It has also been shown that in some tumour types, macrophages appear to mediate the effect of adenosine more potently than T cells[10]. \\u003c/p\\u003e\\n\\u003cp\\u003eWe hypothesised that adenosine would polarise human macrophages towards an immunosuppressive phenotype via both the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors, and that dual receptor blockade would be required to reverse this. Here we present evidence that dual A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor antagonism is required to reverse the effects of NECA on macrophages and prevent deleterious downstream effects on cells important for anti-tumour immunity. \\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003eReagents:\\u003c/p\\u003e\\n\\u003cp\\u003eEndotoxin free reagents were used throughout. \\u0026nbsp;All media were based on RPMI 1640 with L-glutamine (Sigma). Macrophage medium contained v/v 10% FCS and 1% sodium pyruvate (Sigma), T cell medium v/v 10% FCS, 1% HEPES, 1% sodium pyruvate, 1% non-essential amino acids and 20µM 2-mercaptoethanol (all Sigma). Cytokines were recombinant human (rh) Granulocyte-macrophage colony-stimulating factor (GM-CSF, 20U/ml, Peprotech), rh Macrophage colony-stimulating factor (M-CSF, 10ng/ml, Immunotools), lipopolysaccharide (LPS, 1mg/ml, Sigma), interferon gamma (IFNγ, 1000U/ml, R\\u0026amp;D). NECA (5′-(N-Ethylcarboxamido)- adenosine), SCH442416 and PSB603 were all from Tocris. Phytohaemaglutinin (PHA, 4μg/ml, Sigma).\\u003c/p\\u003e\\n\\u003cp\\u003eMacrophage generation:\\u003c/p\\u003e\\n\\u003cp\\u003ePBMC were separated from heparinised blood immediately after venepuncture by density centrifugation over Histopaque 1077 (Sigma). \\u0026nbsp;CD14+ cells were isolated using Miltenyi CD14+ microbead (purity \\u0026gt;95% by flow cytometry). CD14+ monocytes were resuspended at 1x10\\u003csup\\u003e6\\u003c/sup\\u003e/ml and plated in ultra-low attachment plates (Corning costar) with either 5x10\\u003csup\\u003e6\\u003c/sup\\u003e/well (6 well plate) or 1x10\\u003csup\\u003e6\\u003c/sup\\u003e/well (24 well plate). For ‘M1’-type macrophages, GM-CSF was added at day 0 at 20U/ml and for ‘M2’-type macrophages, M-CSF was added at day 0 at 10ng/ml. Macrophages were fed at day 4 with equal volume medium + GM-CSF or M-CSF as appropriate. In some experiments, NECA, SCH442416 and/or PSB603 (all 1μM) were added to macrophage medium at d0 and d4 of differentiation. On day 7, macrophages were harvested by incubation on ice for 15 minutes in cold endotoxin-free PBS (Sigma), resuspended in macrophage medium (or T cell medium for co-culture experiments), plated and rested for 2 hours at 37\\u003csup\\u003eo\\u003c/sup\\u003e/5%CO\\u003csub\\u003e2\\u003c/sub\\u003e prior to any drugs/stimulation.\\u003c/p\\u003e\\n\\u003cp\\u003eIn vitro stimulation:\\u003c/p\\u003e\\n\\u003cp\\u003eMacrophages were resuspended at 5x10\\u003csup\\u003e5\\u003c/sup\\u003e/ml and plated at 5x10\\u003csup\\u003e4\\u003c/sup\\u003e per well in a standard 96-well plate. After resting for 2 hours macrophages were stimulated with LPS (1mg/ml) + IFNγ (1000U/ml) (GM-CSF macrophages) or LPS alone (M-CSF macrophages). In some experiments, NECA (1nM-10μM) and/or adenosine receptor inhibitor drugs (1μM) were added for 1 hour prior to stimulation. After 24hrs, supernatants were harvested for ELISA for IL-12p70 (BD Biosciences) and IL-10 (R\\u0026amp;D) or for multiplex analysis using a custom-designed Luminex Human Magnetic Assay (R\\u0026amp;D Systems).\\u003c/p\\u003e\\n\\u003cp\\u003ePBL:macrophage co-culture:\\u003c/p\\u003e\\n\\u003cp\\u003eAllogeneic peripheral blood leucocyte (PBL, CD14- fraction from monocyte isolations, frozen immediately after isolation at 2x10\\u003csup\\u003e7\\u003c/sup\\u003e/ml in 90% FCS + 10% DMSO) were defrosted and rested overnight at 2x10\\u003csup\\u003e6\\u003c/sup\\u003e/ml in T cell medium in 6 well plates (Costar). The next day, day 7 macrophages were harvested, plated at 1x10\\u003csup\\u003e3\\u003c/sup\\u003e, 3.3x10\\u003csup\\u003e3\\u003c/sup\\u003e, 1x10\\u003csup\\u003e4\\u003c/sup\\u003e or 2x10\\u003csup\\u003e4\\u003c/sup\\u003e/well in 100μl T cell medium in a 96-well plate and rested for 2 hours at 37\\u003csup\\u003eo\\u003c/sup\\u003e/5%CO\\u003csub\\u003e2. \\u0026nbsp;\\u003c/sub\\u003ePBL were then harvested, counted by haemocytometer, resuspended and added at 1x10\\u003csup\\u003e5\\u003c/sup\\u003e per well. PHA was added at 4μg/ml in a final volume of 200μl. On day 4, 50μl supernatant was removed for ELISA for IFNγ (BD Biosciences), IL-10 and IL-17 (both R\\u0026amp;D). 50μl T cell medium containing \\u003csup\\u003e3\\u003c/sup\\u003eH-Thymidine was added immediately and cells cultured for a further 18hr then frozen at -20°C. Cells were subsequently transferred to 96-well unifilter microplates (PerkinElmer), dried overnight at room temperature then Microscint-O (PerkinElmer) added and read on a TopCount NXT (PerkinElmer). PBL without PHA stimulation were used as negative control and had undetectable levels of all cytokines and negligible thymidine incorporation (not shown).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eStatistics\\u003c/p\\u003e\\n\\u003cp\\u003eData were analysed using Graphpad Prism with tests as indicated in figure legends. Summary data for multiple donors was normalised where appropriate to control condition for each donor then mean +/- SEM calculated for all donors together. In all figures, *=p\\u0026lt;0.05, **=p\\u0026lt;0.005 , ***=p\\u0026lt;0.001.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eNECA modulates the function of both fully differentiated and differentiating human macrophages\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur first aim was to confirm the effect of the adenosine analogue NECA on both fully differentiated and differentiating human macrophages. We used NECA throughout this study due to its longer half-life for \\u003cem\\u003ein vitro\\u003c/em\\u003e experiments. To study the effect of NECA on fully differentiated macrophages, CD14+ monocytes were differentiated in medium containing either GM-CSF (to model an \\u0026lsquo;M1\\u0026rsquo; phenotype) or M-CSF (to model an \\u0026lsquo;M2\\u0026rsquo; phenotype). Macrophages were stimulated with LPS/IFN\\u0026gamma; or LPS respectively to readout on their IL-12 and IL-10 cytokine production as a measure of their pro- or anti-inflammatory capacity. GM-CSF differentiated macrophages (GMCSF-M\\u0026phi;) produced abundant IL-12 and smaller amounts of IL-10, whilst M-CSF differentiated macrophages (MCSF-M\\u0026phi;) produced little/no IL-12 and higher levels of IL-10 (Figure 1a).\\u003c/p\\u003e\\n\\u003cp\\u003eTreatment with NECA for 1 hour prior to activation led to a significant concentration-dependent reduction in IL-12 production by GMCSF-M\\u0026phi; and no significant changes in IL-10 (Figure 1bi, p\\u0026lt;0.0001 and p=0.66 by one-way ANOVA respectively). For MCSF-M\\u0026phi;, NECA surprisingly but reproducibly (and in contrast to murine data) produced a significant concentration-dependent decrease in IL-10 (Figure 1bii, p\\u0026lt;0.0001 by one-way ANOVA).\\u003c/p\\u003e\\n\\u003cp\\u003eThe A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor has been shown to be the most important adenosine receptor for mediating adenosine effects on T cells[16] and is the main current clinical target within the adenosine receptor family. In contrast, both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors are known to be expressed in murine and human myeloid cells[26-28]. We therefore used selective small molecule inhibitors of the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors (SCH442416 and PSB603 respectively, both 1\\u0026mu;M) to investigate which adenosine receptor was mediating the effect of NECA in human macrophages. Treatment of macrophages with A\\u003csub\\u003e2A\\u003c/sub\\u003e or A\\u003csub\\u003e2B\\u003c/sub\\u003e antagonist alone did not result in any change in cytokine production (not shown). In GMCSF-M\\u0026phi;, blockade of the A\\u003csub\\u003e2A\\u003c/sub\\u003e or A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor alone prior to NECA treatment and activation produced no significant recovery in IL-12 production (Figure 1c left panel, top and middle). However, simultaneous blockade of both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors was required to significantly recover IL-12 secretion (Figure 1C left panel, bottom, p\\u0026lt;0.001 at NECA concentrations of 100nM and above). Whilst the small increase in IL-10 secretion by NECA-treatment of GMCSF-M\\u0026phi; did not reach statistical significance (Figure 1bi), there was a significant reduction in IL-10 following dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade (Figure 1c middle panel, bottom). In MCSF-M\\u0026phi;, the effect of NECA on IL-10 production was largely reversed by blockade of A\\u003csub\\u003e2B\\u003c/sub\\u003e alone (Figure 1c right panel, middle, p\\u0026lt;0.001 at NECA concentrations of 1\\u0026mu;M and above) whilst A\\u003csub\\u003e2A\\u003c/sub\\u003e blockade alone had very little effect (Figure 1c right panel, top). Thus, the \\u0026lsquo;M2\\u0026rsquo; polarising effect of NECA on differentiated GMCSF-M\\u0026phi; was only completely reversed by blockade of both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors, but blockade of A\\u003csub\\u003e2B\\u003c/sub\\u003e alone largely reversed the effect of NECA on differentiated MCSF-M\\u0026phi;.\\u003c/p\\u003e\\n\\u003cp\\u003eIn the TME, monocytes exiting the vasculature and entering the tumour would be instantly exposed to higher levels of adenosine. Therefore, we studied the effect of NECA (1\\u0026mu;M) on the differentiation of macrophages in order to more accurately mimic conditions experienced in the TME. Similar to the effects of NECA treatment after differentiation, the presence of NECA during differentiation significantly suppressed production of IL-12 from GMCSF-M\\u0026phi; (Figure 2a). In contrast to NECA-treatment after differentiation, IL-10-secreting capacity was substantially increased when NECA was present during differentiation in both GMCSF-M\\u0026phi; and MCSF-M\\u0026phi;, in keeping with previous murine data (Figure 2a). This resulted in a significant reduction in the IL-12:IL-10 ratio in NECA-conditioned GMCSF-M\\u0026phi; (Figure 2bi, p\\u0026lt;0.05) and a mean 2.10-fold increase in IL-10 production (p\\u0026lt;0.05, Figure 2bii).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDual blockade of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors is required to reverse the effect of adenosine on differentiating human macrophages\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHaving established that both the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors mediate the effects of adenosine on fully differentiated human macrophages, we used the small molecule inhibitors during differentiation to establish which receptors were responsible for the repolarisation effect seen. There was no significant effect of adenosine receptor blockade alone on macrophage differentiation (not shown). Once again, combined A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade was required to recover IL-12 production by NECA-conditioned GMCSF-M\\u0026phi; (Figure 2ci, p\\u0026lt;0.005). Notably, single A\\u003csub\\u003e2A\\u003c/sub\\u003e or A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade did not recover IL-12 production in GMCSF-M\\u0026phi; (Figure 2ci). \\u003c/p\\u003e\\n\\u003cp\\u003eIn MCSF-M\\u0026phi;, where NECA-conditioning significantly increased IL-10 production, combined A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade significantly reduced IL-10 production (Figure 2cii). \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNECA has widespread effects on human macrophage function, mediated via both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWhilst IL-12 and IL-10 are an effective readout of \\u0026lsquo;M1\\u0026rsquo; vs \\u0026lsquo;M2\\u0026rsquo;-type macrophages, many other cytokines and chemokines are produced which are important in a tumour context. We therefore wanted to establish what other effects NECA-conditioning had on macrophages and determine whether these were also mediated via both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors. We carried out a multiplex analysis of other cytokines and chemokines produced by macrophages stimulated after differentiation in the presence of NECA (1\\u0026mu;M). Baseline comparison between control GM- M\\u0026phi; and M- M\\u0026phi; is shown in Supplementary Figure 1 and shows that, as expected, GM- M\\u0026phi; produce more pro-inflammatory cytokines (IL-23, IL1\\u0026beta;, IL-6 and TNF\\u0026alpha;) whilst M- M\\u0026phi; produce more MMP7, MMP9 and VEGF-A. In M- M\\u0026phi;, which are more representative of TAMs, we saw several marked and significant changes in cytokine/chemokine secretion after NECA-differentiation. IL-23, CCL-22, IL-6, TNF\\u0026alpha; , FGF-b, IL-1\\u0026beta; and MMP2 were all significantly reduced in NECA-differentiated MCSF-M\\u0026phi; (Figure 3). For the majority, significant recovery was only seen with dual A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade (SCH442416 and PSB603, 1\\u0026mu;M each). VEGF-A was markedly induced by NECA-differentiation in M- M\\u0026phi; and again only fully reversed by dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade. There was no significant effect on VEGF-C or VEGF-D (not shown). MMP2 production by NECA-differentiated M- M\\u0026phi; was also significantly increased. Notably, GMCSF-M\\u0026phi; were affected much less by NECA-polarisation than M- M\\u0026phi;, with significant alterations only seen in Eotaxin 2 and CCL17 (Supplementary Figure 2).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNECA-induced repolarisation of human macrophages impacts on downstream T cell function\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn order to determine whether NECA-polarisation of macrophages has a significant downstream impact on the effector immune cells which can attack and eliminate tumours, we carried out macrophage-PBL co-cultures. NECA was not included in the co-culture in order to assess the impact of the NECA-conditioned macrophages alone. IFN\\u0026gamma;, IL-10 and IL-17 were measured to assess T cell polarisation. NECA-conditioned MCSF-M\\u0026phi; significantly suppressed the ability of PBL to secrete IFN\\u0026gamma; compared to control MCSF-M\\u0026phi; (Figure 4ai (representative donor) and biii, p\\u0026lt; 0.05). This was a dose-dependent effect (Figure 4b and supplementary figure 3) and was reversed by presence of A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e antagonism during macrophage differentiation (Figure 4ai and aiii, p=0.0410 and supplementary figure 3). IL-10 and IL-17 were not affected in MCSF-M\\u0026phi; (Figure 4ai and iii). In keeping with the more modest effects seen on other cytokines and chemokines, NECA-conditioning of GMCSF-M\\u0026phi; did not affect PBL IFN\\u0026gamma; production (Figure 4ai and aii). Stimulation of A\\u003csub\\u003e2A\\u003c/sub\\u003e receptors in T cells suppresses their ability to proliferate[32] so a potential mechanism for this was a reduced absolute number of PBL due to carry-over of NECA. However, we demonstrated that NECA-conditioned macrophages did not alter PBL proliferation compared to control macrophages (Figure 4ci \\u0026amp; ii), confirming that the reduced IFN\\u0026gamma; production was due to alterations in PBL phenotype in response to NECA-differentiated macrophages.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eOur data show that dual A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor antagonism is required to completely reverse the effects of NECA on human macrophages. Furthermore, we demonstrate that the cytokine and chemokine alterations induced by NECA during macrophage differentiation have significant critical downstream effects on the adaptive immune cells which are required for tumour immunotherapy to be effective. The fact that these cytokine and chemokine changes were only reversed by dual A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor blockade indicates that in tumours where there is a significant macrophage infiltration, A\\u003csub\\u003e2A\\u003c/sub\\u003e blockade alone will not be sufficient to repolarise those macrophages to support tumour immunotherapy. In murine macrophages, it has been demonstrated using A\\u003csub\\u003e2A\\u003c/sub\\u003e-receptor knockout studies that adenosine signalling via the A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor alone can suppress TNFα production[27]. However, in wild-type mice with expression of both A\\u003csub\\u003e2A \\u003c/sub\\u003eand A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors, an A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor antagonist alone could not block the effects of adenosine, in keeping with our data. Another group also demonstrated in murine macrophage models that both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e could mediate effects of adenosine when the other was not present[33]. In human lung macrophages (from patients undergoing lung resection for lung carcinoma, but taken from sections of lung distant to the tumour), a reduction in TNFα in response to NECA was seen[26]. Notably, whilst some reversal of this effect was seen with A\\u003csub\\u003e2A\\u003c/sub\\u003e antagonism, it was not complete. A further paper on human monocytes also demonstrated partial recovery of TNFα production by human monocytes when NECA-treated in the presence of an A\\u003csub\\u003e2A\\u003c/sub\\u003e antagonist alone [30]. However, crucially, none of these studies nor any others have examined the relative importance of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors when physiologically expressed and acting together in either murine or human macrophages. Our work is therefore the first to conclusively show that the effect of adenosine is mediated via both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors in human macrophages. \\u003c/p\\u003e\\n\\u003cp\\u003eIt has been shown that when A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors are co-expressed, the lower-affinity A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor tends to dictate the response to both NECA and adenosine[34, 35]. For example, in HEK 293G cells, the A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor contribution to cAMP responses to adenosine and NECA is very small, and the effect of A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor antagonism is limited[35]. Small signals can, however, be observed with selective A2A agonists[35]. We have generated both A\\u003csub\\u003e2A\\u003c/sub\\u003e- and A\\u003csub\\u003e2B\\u003c/sub\\u003e-specific fluorescent ligands[36, 37] and are characterising the relative levels of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e in human macrophages generated under a variety of conditions. It is possible that different relative levels of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors in our GMCSF-Mφ and MCSF-Mφ would explain some of the differences in response to single antagonist treatment. Interestingly, in human lung macrophages it has been shown that A\\u003csub\\u003e2B\\u003c/sub\\u003e expression is higher than A\\u003csub\\u003e2A\\u003c/sub\\u003e in the resting state[26]. The majority of clinical trials in cancer targeting adenosine receptors are focussed on the A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor in combination with checkpoint inhibition. Further work is now needed to explore in relevant pre-clinical models the additional benefit of dual A2A/A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade in combination with checkpoint inhibition over checkpoint + A2A blockade alone. There is a dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e antagonist, AB928 for which phase 1b/2 studies are ongoing and showing encouraging efficacy[38]. Another compound targeting the A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor alone is currently in trials for non-small cell lung cancer after demonstration that A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade in combination with anti-PD1 was more beneficial than anti-PD1 alone[39]. A murine paper shows that combination of caffeine (a non-specific adenosine receptor antagonist) with anti-PD1 therapy leads to better tumour control in a carcinogen-induced tumour model and the B16F10 model than either therapy alone, with accompanying reduction in Treg infiltration and increase in both CD4 and CD8 cells in the tumour[40]. Another paper, whilst showing that caffeine was beneficial when combined with T-cell therapy, showed more substantial benefits with specific pharmacological inhibition of A\\u003csub\\u003e2A\\u003c/sub\\u003e alone[41]. This suggests that whilst dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade has promise, specific and high affinity pharmacological targeting will be required to realise the full potential of this approach. The latter paper was predominantly exploring A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor knockout/blockade in T cells but does also contain data supporting the concept that dual knockout of A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e even in T cells has an additional benefit over A\\u003csub\\u003e2A\\u003c/sub\\u003e blockade alone[41]. In addition to the role of A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors on immune cells, there is renewed interest in this receptor more broadly within tumours as a potential therapeutic target[42]. Furthermore, in a study investigating T cell-adenosine interactions, whilst T-cell A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor expression was generally higher than A\\u003csub\\u003e2B\\u003c/sub\\u003e, it is notable that in T cell populations that expressed higher than normal A\\u003csub\\u003e2B\\u003c/sub\\u003e levels, A\\u003csub\\u003e2B\\u003c/sub\\u003e inhibition could reverse adenosine-mediated suppression of effector function equally effectively as A2A inhibition[15]. Overall, our data and these other studies support the concept that dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade may be required for cancer immunotherapy rather than targeting the A\\u003csub\\u003e2A\\u003c/sub\\u003e receptor alone.\\u003c/p\\u003e\\n\\u003cp\\u003eOur multiplex analysis of secreted cytokines and chemokines shows that NECA has a much more marked effect on MCSF-Mφ than GMCSF-Mφ. GM-CSF is a strongly pro-inflammatory cytokine, and appears to ‘protect’ the macrophages from polarisation towards an M2 phenotype. Given that MCSF-Mφ are more representative of TAMs, the very marked effect of NECA seen on multiple secreted factors in this type of macrophage confirms the importance of developing methods to block the action of adenosine on macrophages in the TME. Whilst several of the cytokines and chemokines altered by NECA were T-cell polarising cytokines (IL-10, TNFα, IL-6 and IL-23), one of the most marked changes was in VEGF-A. It is unsurprising that NECA induces macrophages to produce more VEGF-A, since adenosine is upregulated in hypoxic environments and stimulation of angiogenesis is a mechanism to reverse hypoxia caused by inadequate blood supply. Previous work also noted an increase in VEGF production by human macrophages treated with adenosine[31]. They studied the impact of single adenosine receptor blockade and saw a significant reduction with A\\u003csub\\u003e2A\\u003c/sub\\u003e blockade alone, however the combination of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e blockade was not examined. Another study showed that in a murine model of oxygen-induced retinopathy, macrophage-specific \\u003cem\\u003eAdora2a\\u003c/em\\u003e deletion did not reduce pathological neovascularisation[43]. One interpretation is that macrophages are not involved in this pathogenesis, but another is that adenosine also acts via A\\u003csub\\u003e2B\\u003c/sub\\u003e in those macrophages and therefore deletion of only A\\u003csub\\u003e2A\\u003c/sub\\u003e would not block its effects. In support of this hypothesis, there is evidence that the A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor is the predominant adenosine receptor controlling VEGF production[44]. CCL22, which was significantly reduced by NECA-conditioning, induces chemotaxis of T cells by binding to the CCR4 receptor. Decreased levels of CCL22 produced by TAMs exposed to high levels of adenosine would therefore impair migration of T cells into the tumour, which would be detrimental to current tumour immunotherapeutic effects. This multi-parameter effect of NECA on human macrophages underscores the importance of targeting adenosine receptors on TAMs to support immunotherapy. \\u003c/p\\u003e\\n\\u003cp\\u003eWe saw a differential effect of NECA-conditioning during differentiation versus NECA treatment after differentiation of macrophages, suggesting that receptor expression and/or function varies in different types of myeloid cells. Our finding that IL-10 decreased with NECA-treatment in fully differentiated MCSF-Mφ was unexpected. Another paper has also noted a decrease in IL-10 when monocytes were treated with the adenosine analogue NECA immediately prior to stimulation[45]. As well as differential effects in different types of myeloid cells, it is likely that other cell types or culture conditions can modulate the effects of adenosine on human macrophages. For this reason, we are working to extend these findings into multi-cellular co-culture systems and explore the effects of other factors which are commonly encountered within the TME or during anti-cancer treatment, including hypoxia, extracellular matrix and radiotherapy. Hypoxia is particularly relevant in this context, given published evidence that A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors are preferentially upregulated in myeloid cells exposed to hypoxia[28].\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, this work is the first to clearly demonstrate that the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e receptors are both non-redundant in mediating the effect of the adenosine analogue NECA on differentiating human macrophages, altering their functional phenotype in a way that modulates downstream immune responses. Dual targeting of A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e should therefore be explored further as a therapeutic strategy for tumour immunotherapy.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eATP: adenosine triphosphate\\u003c/p\\u003e\\n\\u003cp\\u003eGM-CSF: Granulocyte-macrophage colony-stimulating factor\\u003c/p\\u003e\\n\\u003cp\\u003eGMCSF-M\\u0026phi;: GM-CSF differentiated macrophages\\u003c/p\\u003e\\n\\u003cp\\u003eIFN: Interferon\\u003c/p\\u003e\\n\\u003cp\\u003eIL: Interleukin\\u003c/p\\u003e\\n\\u003cp\\u003eLPS: Lipopolysaccharide\\u003c/p\\u003e\\n\\u003cp\\u003eM-CSF: Macrophage colony-stimulating factor\\u003c/p\\u003e\\n\\u003cp\\u003eM\\u0026phi;: macrophage\\u003c/p\\u003e\\n\\u003cp\\u003eMCSF-M\\u0026phi;: M-CSF differentiated macrophages\\u003c/p\\u003e\\n\\u003cp\\u003eNECA: 5\\u0026prime;-(N-Ethylcarboxamido)- adenosine\\u003c/p\\u003e\\n\\u003cp\\u003ePBL: Peripheral blood leucocytes\\u003c/p\\u003e\\n\\u003cp\\u003ePHA: Phytohaemaglutinin\\u003c/p\\u003e\\n\\u003cp\\u003eTAM: Tumour-associated macrophage\\u003c/p\\u003e\\n\\u003cp\\u003eTME: Tumour microenvironment\\u003c/p\\u003e\\n\\u003cp\\u003eTNF: Tumour-necrosis factor\\u003c/p\\u003e\\n\\u003cp\\u003eVEGF: Vascular endothelial growth factor\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by a Cancer Research UK Postdoctoral Research Bursary for Clinicians (HF C50808/A24952), by the Medical Research Council (grant no. MR/W016176/1) and by the University of Nottingham. HF was an NIHR-funded Academic Clinical Lecturer in Medical Oncology.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHF has received speaker’s honoraria from L’Oreal. None of the authors have a conflict of interest for this work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions:\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceived project: HF, SH; Designed experiments: HF, SH, AJ, PP, AM, TM; Carried out experiments: HF, FP, AM, BH, AK; Analysed data: HF, FP, BH, AK, TM; \\u0026nbsp; Wrote manuscript: HF; Reviewed manuscript: FP, AM, BH, TM, AK, PP, SH, AJ, HF\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement:\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll donors were recruited in accordance with Good Clinical Practice and the Declaration of Helsinki and provided written informed consent. \\u0026nbsp;Ethical approval was provided by University of Nottingham Medical School Ethics Committee (ref 161-1711).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eWolchok, J.D., et al., \\u003cem\\u003eFinal, 10-Year Outcomes with Nivolumab plus Ipilimumab in Advanced Melanoma.\\u003c/em\\u003e N Engl J Med, 2024.\\u003c/li\\u003e\\n\\u003cli\\u003eCaux, C., \\u003cem\\u003eA Milestone Review on How Macrophages Affect Tumor Growth.\\u003c/em\\u003e Cancer Research, 2016.\\u003c/li\\u003e\\n\\u003cli\\u003eBlay, J., T.D. 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Nayak, \\u003cem\\u003eCaffeine-enhanced anti-tumor activity of anti-PD1 monoclonal antibody.\\u003c/em\\u003e Int Immunopharmacol, 2019. \\u003cstrong\\u003e77\\u003c/strong\\u003e: p. 106002.\\u003c/li\\u003e\\n\\u003cli\\u003eKjaergaard, J., et al., \\u003cem\\u003eA2A Adenosine Receptor Gene Deletion or Synthetic A2A Antagonist Liberate Tumor-Reactive CD8(+) T Cells from Tumor-Induced Immunosuppression.\\u003c/em\\u003e J Immunol, 2018.\\u003c/li\\u003e\\n\\u003cli\\u003eStrickland, L.N., et al., \\u003cem\\u003eThe resurgence of the Adora2b receptor as an immunotherapeutic target in pancreatic cancer.\\u003c/em\\u003e Front Immunol, 2023. \\u003cstrong\\u003e14\\u003c/strong\\u003e: p. 1163585.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Z., et al., \\u003cem\\u003eEndothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis.\\u003c/em\\u003e Nat Commun, 2017. \\u003cstrong\\u003e8\\u003c/strong\\u003e(1): p. 584.\\u003c/li\\u003e\\n\\u003cli\\u003eRyzhov, S., et al., \\u003cem\\u003eRole of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.\\u003c/em\\u003e Mol Pharmacol, 2014. \\u003cstrong\\u003e85\\u003c/strong\\u003e(1): p. 62-73.\\u003c/li\\u003e\\n\\u003cli\\u003eLe Moine, O., et al., \\u003cem\\u003eAdenosine enhances IL-10 secretion by human monocytes.\\u003c/em\\u003e J Immunol, 1996. \\u003cstrong\\u003e156\\u003c/strong\\u003e(11): p. 4408-14.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Macrophage, adenosine, receptor, human, cancer immunotherapy\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6975438/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6975438/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eAdenosine receptors are a target for cancer immunotherapy, with high levels of adenosine in the tumour microenvironment producing immunosuppressive effects. Most clinical trials in cancer are targeting the A\\u003csub\\u003e2A\\u003c/sub\\u003e adenosine receptor, but evidence suggests that both the A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B \\u003c/sub\\u003ereceptors are important for determining the effect of adenosine in myeloid-lineage cells. We therefore studied the adenosine receptors involved in mediating the effect of the adenosine analogue 5'-\\u003cem\\u003eN\\u003c/em\\u003e-Ethylcarboxamidoadenosine (NECA) on human monocyte-derived macrophages and assessed the down-stream effect of NECA-conditioned macrophages on T cell function. NECA-conditioning of human macrophages led to changes in cytokine and chemokine production resulting in a more ‘M2’ phenotype (decreased IL-12, IL-23, IL-6, TNFa and increased VEGF-A and IL-10). NECA-conditioned macrophages altered T cell phenotype in co-culture, impairing IFNγ production. Dual blockade of both A\\u003csub\\u003e2A\\u003c/sub\\u003e and A\\u003csub\\u003e2B\\u003c/sub\\u003e adenosine receptors was required to reverse the cytokine and chemokine changes seen in NECA-conditioned macrophages and to recover T cell IFNγ production. These data indicate that dual A\\u003csub\\u003e2A\\u003c/sub\\u003e/A\\u003csub\\u003e2B\\u003c/sub\\u003e receptor blockade will be required to re-polarise macrophages in a tumour environment to support cancer immunotherapeutic approaches.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Dual blockade of adenosine A2A and A 2B receptors is required to reverse NECA-induced immunosuppression in human macrophages: Implications for cancer immunotherapy\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-09 15:22:19\",\"doi\":\"10.21203/rs.3.rs-6975438/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f9fb0a1d-d01c-4dd3-93f2-a24996acb142\",\"owner\":[],\"postedDate\":\"September 9th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-09T15:22:19+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-09-09 15:22:19\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6975438\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6975438\",\"identity\":\"rs-6975438\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}