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A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

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A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells
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  A Novel Copper Chelate Modulates Tumor AssociatedMacrophages to Promote Anti-Tumor Response of T Cells Shilpak Chatterjee 1 . , Ananda Mookerjee 3 . , Jayati Mookerjee Basu 3 . , Paramita Chakraborty 1 , Avishek Ganguly 1 , Arghya Adhikary 4 , Debanjan Mukhopadhyay 5 , Sudipta Ganguli 5 , Rajdeep Banerjee 6 ,Mohammad Ashraf  2 , Jaydip Biswas 2 , Pradeep K. Das 6 , Gourisankar Sa 4 , Mitali Chatterjee 5 , Tanya Das 4 ,Soumitra Kumar Choudhuri 1 * 1 Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, India,  2 Department of Surgical Oncology, HospitalUnit, Chittaranjan National Cancer Institute, Kolkata, India,  3 INSERM, U-563, CHU Purpan, Toulouse, France,  4 Department of Molecular Medicine, Bose Institute, Kolkata,India,  5 Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India,  6 Rajendra Memorial Research Institute of MedicalSciences, Patna, India Abstract Background:   At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs)might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis. Methodology/Principal Findings:   The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG),synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistantcancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive toproimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10that not only allowed strong Th1 response marked by generation of high levels of IFN- c  but also reduced activation inducedT cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractorycancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogrammingof TGF- b  producing CD4 + CD25 + T cells toward IFN- c  producing T cells. Conclusion/Significance:   Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancersthrough reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth. Citation:  Chatterjee S, Mookerjee A, Mookerjee Basu J, Chakraborty P, Ganguly A, et al. (2009) A Novel Copper Chelate Modulates Tumor AssociatedMacrophages to Promote Anti-Tumor Response of T Cells. PLoS ONE 4(9): e7048. doi:10.1371/journal.pone.0007048 Editor:  Benjamin Edward Rich, Harvard Institute of Medicine, United States of America Received  February 20, 2009;  Accepted  August 18, 2009;  Published  September 16, 2009 Copyright:    2009 Choudhuri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  Indian council of Medical research, New Delhi, India; WWW.icmr.nic.in Grant Number: 5/13/18/2007 NCDIII and 5/13/18/2004 NCDIII. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: soumitra01@yahoo.com .  These authors contributed equally to this work. Introduction Tumor cells escaping from the immune surveillance inimmunocompetent individuals reflects inadequate function of theimmune system. Induction of tumor specific T cell mediatedimmunity may block the tumor growth and may give protectiveanti-tumor immune response [1,2]. However strong immunesuppression in the tumor microenvironment makes the situationmore complicated [3,4]. It has been suggested that the growing tumors produce various chemoattractants that have been impli-cated in recruitment of monocytes in the tumor site. Whenmonocytes are recruited into the growing tumor site, local cytokinemilieu modulates the immunological functions of these newlyrecruited monocytes and educates them towards tumor-associatedmacrophages (TAMs) that are immunosuppressive in nature[5,6,7]. TAMs promote tumor cell proliferation and metastasisby secreting a wide range of growth and proangiogenic factors aswell as various metalloproteinases [8,9]. TAMs also possess poorantigen presenting ability and effectively suppress the induction of proper anti-tumor T cell response through the production of immunosuppressive cytokines like TGF- b  and IL-10 [6,10], as wellas promote induction and infiltration of CD4 + CD25 + FoxP3 + Tcells (Treg) at the tumor site [11]. However, evidence suggestedthat phenotype of TAMs can be reprogrammed and the presenceof IL-12 in its local milieu plays key role in reprogramming of theirfunctional cytokine profile towards proimmunogenic (IL-12secreting) nature [12]. IL-12 also dictates the orchestration of Tcell response towards generation of protective anti tumor responseby stimulating T cells and NK cells to produce IFN-  c  (13, 14, 15).Induction of IFN-  c  production and suppression of IL-4production by IL-12 has been shown to induce anti-tumorresponse in murine tumor models [14,16]. Thus, if TAMs possess PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e7048  functional plasticity, it would be useful target for anti-tumortherapy because skewing them again towards proimmunogenicnature could induce proper anti tumor Th1 response that caneffectively reduce tumor growth and metastasis.It has been reported earlier that copper homeostasis plays a vitalrole in drug resistance in cancer and also found to be essential inmediating several intracellular signals in macrophage [17,18]. Anelevated copper level in macrophage is associated with theproduction of inflammatory cytokines whereas a copper deficiencyattenuates its conventional immunological functions [19]. Previ-ously our laboratory had synthesized a novel copper chelate[Copper N-2(hydroxy acetophenon) glycinate (CuNG)] which wasfound to be a potent immunomodulator able to elevate thenumber of CD4 + IFN- c  producing cells in drug resistant tumor[Doxorubicin resistant Ehrlich Ascites Carcinoma (EAC/Dox)]bearing mice [20,21]. In this study we found that CuNG has directeffect on TAMs and can modulate their functional cytokinepattern, inducing their conversion from immunosuppressive toproimmunogenic nature. Herein we have also found that changein regulatory cytokine profile of TAMs was able to redirect the Thelper function, reprogram Treg population and augment theinduction of protective immune response in EAC/Dox bearing mice. Similar results were also obtained in case of peripheral bloodmonocytes from chemo and/or radiotherapy refractory patients. Results CuNG treatment can directly modulate the regulatorycytokine profile of Tumor Associated Macrophages(TAMs) Previous study with the novel copper chelate CuNG revealed itsimmunomodulatory properties in EAC/Dox bearing mice. CuNGcaused augmentation of apoptogenic inflammatory cytokine(mainly IFN- c  ) production and resolution of tumors [21]. Thisfinding prompted us to further investigate how this copper chelate(CuNG) induces the production of such inflammatory cytokines.First we have tried to know whether CuNG can directly act onCD4 + T cells [as this population is the predominant source of inflammatory cytokines following CuNG administration (i.m) [21]]to induce IFN- c  production. It was observed that in vitroapplication of CuNG did not have any significant effect in theIFN- c  production by CD4 + T cells obtained from EAC/Doxbearing mice (Fig. 1A). TAMs play pivotal role in suppression of IFN- c  producing CD4 + T cells (Th1 response) at the tumor sitethrough establishment of immunosuppressive cytokine environ-ment [22]. Therefore, we probed whether CuNG could modulatethe functional behavior of TAMs from suppressive to proimmu-nogenic type so that protective Th1 response can be elicited. Totest this possibility, EAC/Dox bearing mice were treated withCuNG (i.m, 5 mg/kg of body weight), TAMs were isolated 15days following CuNG treatment (i.e., when tumors start to reduceprominently) and intracellular cytokine profile was checked byflow cytometry (Fig. 1B). It was observed that TAMs isolated fromCuNG treated group released elevated level of IL-12 compared tothe TAMs obtained from untreated EAC/Dox bearing mice(77.36% vs. 22.6%, MFI: 71.89 6 1.24 vs. 15.35 6 1.42). On theother hand, production of two major suppressive cytokines, IL-10(63.21% vs. 94.09%, MFI: 48.68 6 0.95 vs. 82.44 6 0.68) and TGF- b  (19.21% vs. 70.68%, MFI: 14.77 6 0.72 vs. 92.35 6 0.81) wasfound to be down regulated in the CuNG treated group comparedto the untreated control.The result was further confirmed by performing ELISA for IL-12, IL-10 and TGF- b . It was observed that 12 h and 24 h culturesof TAMs in the presence of CuNG did not show any modulationin regulatory cytokine production but 48 h of CuNG treatmentcaused significant up regulation in IL-12 production (Fig. 1E)(  . 13.5 fold) whereas the suppressive cytokine TGF- b  (Fig. 1F)production was highly (  , 17.2 folds) and IL-10 production (Fig. 1D)was moderately down regulated (  , 3.71 folds) as compared to theuntreated control. These results together indicate that CuNGtreatment significantly modulates the production of regulatorycytokines by TAMs.Interestingly, it was observed that CuNG treated TAMsmaintain a sustained higher level of reactive oxygen species(ROS; measured in terms of peroxide) till 18 h post treatmentcompared to untreated TAMs (Fig. 1C). Chelation of ROS withthe anti-oxidant tocopherol (50  m M) reversed the nature of CuNGtreated TAMs by increasing IL-10 and TGF- b  production anddecreasing IL-12 generation to levels comparable to untreatedTAMs (Fig. 1D, E and F). In vivo administration of CuNG in EAC/Dox bearing miceinduce Th1 type response It has well been documented that the cytokine IL-12 plays apivotal role in Th1 polarization [13,15]. Since administration (i.m)of CuNG in EAC/Dox bearing mice induce elevated level of IL-12 production by TAMs at the tumor microenvironment, wetherefore checked whether in vivo CuNG treatment couldmodulate the cytokine profile of the tumor associated lymphocytes(TAL) towards Th1 type. Flow cytometric analysis revealed thatCD4 + population of TAL from CuNG treated group showedhigher percentage of IFN- c  positive population compared tountreated group (21.42% vs. 3.54%). On the contrary, thepercentage of IL-4 and TGF- b  positive populations in TALsisolated from in vivo CuNG treated animals was found to begreatly reduced compared to that from untreated animals (7.73% vs. 17.59% and 6.18% vs. 19.24%respectively) (Fig. 2). Soluble factors from functionally altered TAMs can skewunresponsive CD4 + T cells of untreated EAC/Dox micetowards Th1 type The conventional role of macrophage in tumor rejection throughrecognition of tumor antigen and participation in induction of anti-tumor T cell response is changed at the tumor site where it seems toproduce elevated levels of immunosuppressive cytokines like IL-10 andTGF- b  that effectively attenuate the induction of anti tumor response.WehaveshownherethatsingleadministrationofCuNGinEAC/Doxbearing mice was able to alter the functional polarization of TAMsfrom immunosuppressive to proimmunogenic in nature leading toinductionofTh1typeofresponseatthetumorsite.Theseobservationsprompted us to investigate whether the soluble mediators derived fromCuNG treated TAMs are sufficient to redirect the tumor associatedunresponsive CD4 + T cells towards Th1 type in the absence of contactdependent signal. To assess this possibility TAMs were isolated fromuntreated EAC/Dox bearing mice and cultured for 48 h in presenceor absence of CuNG. On the other hand TAMs from in vivo CuNGtreated mice were cultured in absence of CuNG for 48 h. Following completion of incubation, cell free supernatants were obtained, diluted2 folds with fresh medium and used to culture CD4 + cell enrichedTALs.Following96 hofincubation,cellswereharvestedandthelevelsof different Th1 and Th2 cytokine specific mRNA expressions werestudied by semi-quantitative RT-PCR using specific primers. Weobserved that CD4 + T cell populations cultured with cell freesupernatant of either in vitro CuNG treated TAMs or TAMs fromin vivo CuNG treated animals manifested significantly elevated levelsof expression of Th1 specific cytokine mRNA (IFN- c  ) whereas Th2specific (IL-4) and suppressive (TGF- b  ) cytokine mRNA expression Reprogramed TAM Changes TALPLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e7048  levels were undetectable or poorly detectable in these two groups. Onthe contrary, IL-4 and TGF- b  mRNA expressions were found to besignificantly higher in the untreated control group (Fig. 3A & B).Similar results were obtained when Th1 and Th2 specific cytokineproductionbyCD4 + Tcellswereanalyzedbyflowcytometry.CD4 + Tcellpopulationculturedwithcellfreesupernatantderivedfromculturesof either in vitro CuNG treated TAMs or TAMs from in vivo CuNGtreated mice,induced augmented IFN- c production fromCD4 + TALsobtained from untreated EAC/Dox bearing mice. Flow cytometricdata (Fig. 3C) also revealed that culture supernatant obtained from in vitro or in vivo CuNG treated TAMs highly reduced the percentage of TGF- b  and IL-4 producing CD4 + T cells. So both mRNA expressionlevel and intracellular cytokine assay clearly indicate that the pattern of functional cytokine production by a lineage committed CD4 + T cellscan be modulated in response to its local cytokine microenvironmentcreated by the antigen presenting cells (like TAMs). CuNG treated TAMs induce reprogramming of cytokinestatus of CD4 + CD25 + T cells CD4 + CD25 + FoxP3 + (Treg) are well known culprit for creationof immunosuppressive tumor microenvironment via production of high levels of TGF- b  [23,24]. It has previously been shown by usthat CuNG treatment in vivo reduces CD4 + CD25 + FoxP3 + T cellsat the tumor site (21). Since under in vitro condition combination Figure 1. Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs.  A) ELISA. B) Flow cytometry. C)Fluorometric analysis. D, E & F) ELISA. In vitro CuNG treatment (2.5  m g/ml) did not change IFN- c  production from CD4 + T cells of TALs of untreatedEAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice andlabeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF- b  or with specific isotype control Abs. Immunofluorescenceanalysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either keptuntreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different timepoints. Results are presented as mean 6 SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated(2 6 10 6 cells/500  m l). Cells were either kept untreated or pretreated with tocopherol (50  m M) for 1 h. Then the cells were further cultured for 12 h,24 h and 48 h in the presence or absence of CuNG (2.5  m g/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E)and TGF- b  (F) by ELISA and results are presented as mean 6 SE of 3 independent experiments, each experiment having every measurement intriplicate.doi:10.1371/journal.pone.0007048.g001Reprogramed TAM Changes TALPLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e7048  of cytokines produced from CuNG treated TAMs can shift thecytokine profile of CD4 + TALs from TGF- b  producing to IFN- c producing, therefore we tested whether it can reprogram Treg towards Th1 type. For this purpose sorted CD4 + CD25 + population from TALs of untreated EAC/Dox bearing mice werelabeled with CFSE and co-cultured for 96 h with either untreatedTAMs or TAMs treated with CuNG in vitro for 48 h. T cellscultured with untreated TAMs maintained their high TGF- b status while cytokine status of T cells cultured with CuNG treatedTAMs was reprogrammed to low TGF- b and high IFN- c (Fig. 4A).Furthermore, majority of the CD25 + sorted CD4 + cells, whencultured with CuNG treated TAMs, lost their FoxP3 expression vis-a`-vis decreasing TGF- b  production and increasing IFN- c generation (Fig. 4B). These cells also lose the characteristicinhibitory property of Treg on proliferation of T cells (Fig. 4C). Redirection of the tumor associated CD4 + T cells towardsTh1 type can be accounted for by CuNG mediatedaltered level of IL-10 and IL-12 production from TAMs It is well evident from Fig. 1B that CuNG treatment causedalteration in the levels of IL-10 and IL-12 production by TAMsbut did not completely abrogate the IL-10 production. Thisobservation made us curious to further investigate whether thecombination of low level of IL-10 and high level of IL-12 that weobtained with TAMs after CuNG treatment would have the samepotential as IL-12 alone to make the decision for the generation of the Th1 response. To test this hypothesis, CD4 + TAL fromuntreated EAC/Dox bearing mice were cultured in the presenceof either recombinant IL-10 (rIL-10) or IL-12 (rIL-12) alone orwith a combination of rIL-10 and rIL-12 for at least 96 h. Thedoses of rIL-10 and rIL-12 applied corresponded to those obtainedfrom the ELISA data of IL-10 and IL-12 production by eitheruntreated or in vitro CuNG treated TAMs. Both mRNAexpression study (Fig. 5A & B) and intracellular cytokinesproduction assay (Fig. 5C) indicated that CD4 + T cells populationcultured in the presence of 0.35 ng/ml of rIL-10 and 2.7 ng/ml of rIL-12 showed a significant up-regulation in production of IFN- c similar to CD4 + TALs treated with only a single high dose of rIL-12. On the other hand, CD4 + TALs stimulated in the presence of 1.3 ng/ml of rIL-10 and 0.2 ng/ml of rIL-12 (amount similar tothe IL-10 and IL-12 obtained from untreated TAMs), did notshow any remarkable change in cytokine production pattern fromthat we observed in case of single high dose of rIL-10 treated oruntreated control group.Interestingly it is also evident from dot plot analysis of flowcytometry data (Fig. 5C) that the percentage of IFN- c  positive cellsamong CD4 + TALs was higher when cultured in the presence of combination of high rIL-12 and low rIL-10 compared to the highrIL-12 alone (54.42% vs. 36.89%). Interestingly, CuNG treatedTAMs, when fixed with paraformaldehyde could not increaseIFN- c  production or decrease TGF- b  generation in CD4 + TALs.However, when a combination of low rIL-10 and high rIL-12 wasintroduced in this system, TALs were reprogrammed (Fig. 5D).Moreover, co-culture of CuNG treated TAMs and CD4 + TALsdid not significantly increase the level of reprogramming (Fig. 5D).These results indicate that reprogramming of TALs stronglydepends on soluble agents (high IL-12 and low IL-10) releasedfrom reprogrammed TAMs. However, the cause of lowerpercentage of IFN- c  producing TALs following rIL-12 treatmentalone compared to that following treatment with high rIL-12 andlow rIL-10 remained unanswered. To explain this differentialresponse we reasoned that the combination of high rIL-12 and lowrIL-10 might block the death of T cells. Presence of small amount of IL-10 in association with IL-12 delayed the death of Th1 population Several reports are corroborating the fact that high level of IFN- c  produced by the Th1 population mediates its own apoptosis byup-regulating both Fas and Fas-L expression [25,26,27,28].Recently a differential role of IL-10 as an anti-apoptotic mediatorprotecting the mouse intestinal epithelial cells from IFN- c  or TNF- a  mediated apoptosis by diminishing the Fas expression has beenshown [29]. These findings prompted us to investigate whether thepresence of low level of rIL-10 in combination with high rIL-12could prolong the Th1 response by interfering with its self-killing mechanism. To address the issue of involvement of IL-10 inprolonging Th1 response, we cultured CD4 + T cells obtained fromTALs of untreated EAC/Dox bearing mice either with a singlehigh dose or different combination of rIL-10 and/or rIL-12 for 5days. Cell death was quantified by means of PI/Annexin V-FITC.It was observed that rIL-12 alone induced high levels of apoptosiswhile a combination of low rIL-10 and high rIL-12 protectedCD4 + T cells from undergoing apoptosis (Fig. 6A). This apoptoticprocess was found to be associated with caspase 3 activation. Anactive caspase 3 level in each experimental group was representedby the fluorescence intensity of the cleaved fluoregenic AMC Figure 2. TALs of EAC/Dox bearing mice showed Th1 specificresponse after in vivo administration of CuNG.  Flow cytometry.EAC/Dox bearing mice (n=12) were treated with CuNG (5 mg/kg of body weight), i.m., 7 days following inoculation and 15 days after CuNGadministration TALs were isolated from the ascitic fluid of treated anduntreated animals as nonadherent population (method described inmaterial and method section). From isolated TALs, CD4 vs. intracellularIFN- c , IL-4 and TGF- b  production were analyzed by flow cytometry andsignificantly higher percentage of CD4 population of treated groupshowed positive for IFN- c  (marker for Th1 response). A representativeresult is presented here for comparison.doi:10.1371/journal.pone.0007048.g002Reprogramed TAM Changes TALPLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7048  liberated due to cleavage of Ac-DEVD-AMC by active caspase 3.Caspase 3 assay clearly indicates (Fig. 6B) that intensity of activecaspase 3 levels in the high rIL-12 treated group increased muchfaster than high rIL-12 plus low rIL-10 treated group(15.663 6 0.57 vs. 15.107 6 1.06 at 72 h, 21.755 6 0.74 vs.16.090 6 0.61 at 96 h and 27.223 6 0.60 vs. 18.414 6 0.36 at120 h respectively). Addition of cell free supernatant derived from48 h culture of both in vitro CuNG treated TAMs (originallyisolated from untreated animals) and in vivo CuNG treated TAMs(obtained from in vivo CuNG treated animals) in the culture of CD4 + TALs from untreated animals resulted in restricted increaseof caspase 3 activity (14.16 6 0.89 at 72 h, 17.59 6 0.46 at 96 h and20.36 6 0.86 at 120 h and 15.65 6 0.46 at 72 h, 16.64 6 0.28 at96 h and 18.93 6 0.53 at 120 h respectively) while neutralization of IL-10 in corresponding sets resulted in rapid increase of caspase 3activity (16.33 6 0.76 at 72 h, 23.22 6 0.94 at 96 h and 29.65 6 0.71 Figure 3. Culture supernatant of TAMs, treated with CuNG, caused altered cytokines production by TALs.  A) RT-PCR. B) Densitometricanalysis. C) Flow cytometry. CD4 +  T cells were purified from TALs obtained from untreated EAC/Dox bearing mice and cultured for 96 h with cell freesupernatant of TAMs obtained from untreated EAC/Dox bearing mice that were either kept untreated for 48 h or treated with CuNG (48 htreatement) in vitro or with cell free supernatant of TAMs (cultured for 48 h in absence of CuNG) obtained from in vivo CuNG (15 days after treatment,i.e., when tumors start regressing prominently) treated EAC/Dox bearing mice. Cytokine profile was analyzed by semi-quantitative RT-PCR. PurifiedCD4 + population from TALs (derived from untreated EAC/Dox bearing mice) cultured without any treatment were used as untreated control. Aftercompletion of 96 h of incubation equivalent amount of mRNA (2  m g) from TALs of each experimental group was used for RT-PCR analysis andrepresentative data from three independent experiment is presented (A). In all cases GAPDH was used as housekeeping gene control. Densitometryanalysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specificfor Th1 (IFN- c ) or Th2 (IL-4) or suppressive (TGF- b ) production profile in the above mentioned experimental groups were also analyzed by flowcytometry and representative data of three independent experiments is presented here (C).doi:10.1371/journal.pone.0007048.g003Reprogramed TAM Changes TALPLoS ONE | www.plosone.org 5 September 2009 | Volume 4 | Issue 9 | e7048
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