Alterations of tumor microenvironment by nitric oxideimpedes castration-resistant prostate cancer growthHimanshu Aroraa,b,1, Kush Panaraa, Manish Kuchakullaa, Shathiyah Kulandavelub, Kerry L. Burnsteinc,Andrew V. Schallyd,e,f,g,h,1, Joshua M. Hareb,i, and Ranjith Ramasamya,1

aDepartment of Urology, Miller School of Medicine, University of Miami, Miami, FL 33136; bThe Interdisciplinary Stem Cell Institute, Miller School ofMedicine, University of Miami, Miami, FL 33136; cDepartment of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami,Miami, FL 33136; dEndocrine, Polypeptide, and Cancer Institute Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Researchand Education, Miami, FL 33125; eDepartment of Medicine, Division of Hematology/Oncology, Miller School of Medicine, University of Miami, Miami, FL33136; fDepartment of Medicine, Division of Endocrinology, Miller School of Medicine, University of Miami, Miami, FL 33136; gDepartment of Pathology,Miller School of Medicine, University of Miami, Miami, FL 33136; hSylvester Comprehensive Cancer Centre, Miller School of Medicine, University of Miami,Miami, FL 33136; and iDepartment of Medicine, Division of Cardiology, Miller School of Medicine, University of Miami, Miami, FL 33136

Contributed by Andrew V. Schally, August 24, 2018 (sent for review August 3, 2018; reviewed by K. C. Balaji, Vinata B. Lokeshwar, and Rama Soundararajan)

Immune targeted therapy of nitric oxide (NO) synthases are beingconsidered as a potential frontline therapeutic to treat patientsdiagnosed with locally advanced and metastatic prostate cancer.However, the role of NO in castration-resistant prostate cancer(CRPC) is controversial because NO can increase in nitrosative stresswhile simultaneously possessing antiinflammatory properties. Ac-cordingly, we tested the hypothesis that increased NO will lead totumor suppression of CRPC through tumor microenvironment. S-nitrosoglutathione (GSNO), an NO donor, decreased the tumorburden in murine model of CRPC by targeting tumors in a cellnonautonomous manner. GSNO inhibited both the abundance ofantiinflammatory (M2) macrophages and expression of pERK, in-dicating that tumor-associated macrophages activity is influencedby NO. Additionally, GSNO decreased IL-34, indicating suppressionof tumor-associated macrophage differentiation. Cytokine pro-filing of CRPC tumor grafts exposed to GSNO revealed a significantdecrease in expression of G-CSF and M-CSF compared with graftsnot exposed to GSNO. We verified the durability of NO on CRPCtumor suppression by using secondary xenograft murine models.This study validates the significance of NO on inhibition of CRPCtumors through tumor microenvironment (TME). These findingsmay facilitate the development of previously unidentified NO-basedtherapy for CRPC.

nitric oxide | tumor microenvironment | CRPC | tumor-associatedmacrophages | immunotherapy

Prostate cancer is the second most frequent cause of cancer-related deaths in men. Men with prostate cancer that has

recurred after local therapy usually respond to androgen depri-vation therapy (ADT); however, despite this treatment, mostpatients eventually experience progression of the disease within 2 y,a condition known as castration-resistant prostate cancer (CRPC)(1). In trying to understand the causes of this androgen resistancethat develops in CRPC, most research has focused directly on thesplice variants of the androgen receptor (ARVs) (2). However, thetumor microenvironment (TME) has been shown to play a majorrole in tumor progression, yet the response to therapy in othercancer types has been inadequately studied in CRPC (3–5). TME iscomprised of a variety of cell types, including immune cells, fibro-blasts, pericytes, and tumor-associated macrophages (TAMs) (6).TAMs are recruited to tumors from diverse signaling moleculessuch as chemokines (CCL-2 and CCL-5) and cytokines (IL-34 andCSF-1) (7). While the exact mechanism is unknown, TAMs havebeen reported to play a key role in the progression of prostatecancer through the secretion of cytokines, matrix metalloproteinases,and growth factors (8, 9).A key molecule in the regulation of TME interactions is the

ubiquitous nitric oxide (NO) (10–12). We have previouslyestablished the importance of NO in the cardiovascular andimmune systems and in male secondary hypogonadism (13–20).

Others have investigated the tumoricidal implications of NO intherapeutic resistance (21–23), cell survival, proliferation of tumors,inhibition of tumor growth, and reduction in lung metastases (24) inmany cancer types (25). Several NO donors, including S-nitrosothiols,organic nitrates, and Metal-NO complexes, have shown impacts oncancer progression (26, 27). S-nitrosothiols such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) havealso shown promising effects as antineoplastic agents. However, thesestudies have only focused on certain aspects of NO such as its role inboth progrowth and antigrowth effects (28), cellular localization,endogenous expression of the androgen receptor (AR) (29), and ARfunction inactivation by S-nitrosylation (30, 31). In the present study,we document the effect of NO on tumor suppression by targeting theCRPC TME through TAMs.

ResultsIncreased NO Levels Affect Testosterone and Luteinizing Hormone.We have previously shown that mice lacking the S-nitrosoglutathionereductase (GSNOR) gene showed increased nitrosative stress andexhibited secondary hypogonadism (19). Therefore, in this study weexamined if increased NO levels are able to suppress testosterone (T)and luteinizing hormone (LH) in control C57BL/6J mice. For theseexperiments, we administered GSNO (an NO donor) intraperito-neally (10 mg/kg) for 7 d and compared T and LH levels to mice

Significance

This study presents insights into the underexplored areas ofcastration-resistant prostate cancer (CRPC) therapeutics—therole of nitric oxide (NO) in CRPC reduction through its micro-environment. Results of this study provide important informa-tion on the tumor reduction capabilities of increased NO levelsand its mechanistic aspect and demonstrates the potential long-term efficacy of NO on CRPC. An in-depth understanding of howNO affects the tumor microenvironment will allow developmentof chemotherapeutics based on NO for a CRPC cure.

Author contributions: H.A., A.V.S., J.M.H., and R.R. designed research; H.A., K.P., and M.K.performed research; H.A., K.P., S.K., K.L.B., A.V.S., and J.M.H. analyzed data; and H.A.,S.K., A.V.S., J.M.H., and R.R. wrote the paper.

Reviewers: K.C.B., University of Florida; V.B.L., Augusta University; and R.S., The Universityof Texas MD Anderson Cancer Center.

Conflict of interest: J.M.H. discloses a relationship with Vestion Inc. that includes equity,board membership, and consulting. J.M.H. is the Chief Scientific Officer, a compensatedconsultant, and advisory board member for Longeveron and holds equity in Longeveron.J.M.H. is also the coinventor of intellectual property licensed to Longeveron.

Published under the PNAS license.1To whom correspondence may be addressed. Email: hxa287@miami.edu, andrew.schally@va.gov, or ramasamy@miami.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1812704115/-/DCSupplemental.

Published online October 15, 2018.

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administered with phosphate buffer saline (PBS). We found that Tand LH levels were decreased 6× in the mice that received GSNO(Fig. 1A). Further validation was obtained by oral administration ofGSNO (10 mg/400 mL bottle for 5 wk) to C57BL/6J mice, as wefound that T levels were undetectable in mice that received oralGSNO compared with untreated animals (Fig. 1B). To confirm ifincreased NO levels are capable of affecting the hypothalamic-pituitary-gonadal axis to regulate LH and T levels, we checkedGnRHR expression in the brain (Fig. 1C), and our results con-firmed that increased NO levels were capable of reducing theexpression of GnRHR in the brain. Together, these results in-dicated that increased NO levels affect receptors for GnRH, LH,and T through the hypothalamic-pituitary-gonadal axis.

NO Reduces Tumor Burden in the Murine Model of CRPC. We hy-pothesized that increased NO can lead to CRPC tumor suppressionbecause of its ability to affect the hypothalamic-pituitary-gonadalaxis. After castration, 2.5 million 22RV1 cells were xenografted s.c.in each flank of SCID mice. GSNO (10 mg/kg per d i.p.) was ad-ministered to half of the mice (experimental group) and an equalvolume of PBS to the remaining animals. After 14 d of GSNOtreatment, the grafts were harvested (Fig. 2A), and we found that

the overall tumor burden was decreased in mice treated with GSNO(high NO levels) (Fig. 2 B–E), significantly affecting the weight ofmice (Fig. 2C). We evaluated the NO levels in tumors isolated frommice treated with PBS or GSNO using Griess test and confirmed aninverse association between tumor burden and NO levels (Fig. 2F).Tumors from mice that received GSNO treatment showed areaswith less necrosis (Fig. 2G and SI Appendix, Fig. S1). Furthermore,GSNO-treated mice showed a reduced number of Ki-67– positivecells, suggesting a reduced proliferation rate (Fig. 2H).

NO Suppresses Tumor Burden in a Cell Nonautonomous Manner.Mostchemotherapeutic drugs as well as ionizing radiation promoteautophagy in tumor cells (32–35). However, autophagy can haveboth cell autonomous and cell nonautonomous effects and influ-ence the outcome of therapy (35). Therefore, we evaluated whetherthe effects of NO are tumor cell autonomous or nonautonomous.We studied the cell proliferation rate after treating the 22Rv1 cellswith varying concentrations of GSNO in vitro. Interestingly, wefound that in vitro the proliferation of 22Rv1 cells was largely un-affected (Fig. 3A), despite the efficacy of GSNO on tumor burdenin vivo (Fig. 2). Because 22Rv1 cell growth is dependent on con-stitutive signaling of the androgen receptor (AR), we examined theAR signaling markers involved in androgen-induced activation ofendogenous AR, such as PSA and TMPRSS2 (29). Both PSA andTMPRSS2 levels were suppressed by escalating doses of GSNO(Fig. 3B). Taken together, changes in AR signaling without changesin cell proliferation in vitro suggest the effects of NO on 22Rv1 cellsare likely cell nonautonomous.

Cytokine Signature Showed That NO Suppresses TAMs Affecting CRPCTME. The cell nonautonomous effects are related largely to theimmune system (35), TME, and subclone heterogeneity (36).Macrophages are known to increase specific circulating cytokineswith progressive metastasis (37, 38). Therefore, to study themolecular events leading to NO-induced changes, we evaluatedprotein expression of 120 cytokines using the tumors from micethat received GSNO versus PBS (SI Appendix, Fig. S2). Among120 cytokines assayed, there were 26 cytokines (CCL27, CD54,TIMP-1, ACRP30, G-CSF, AR, IL-17A, beta NGF, IL-2 R al-pha, CCL28, Axl, CCL7, CCL17, CXCL6, IL-1 F2, M-CSF,TGFBeta 3, CXCL13, BMP-6, CCL23, NT-3, CCL11, CCL1,IL-5, IL-6, and IGFBP4) that were suppressed more than 1.5 times(Fig. 3C). From previously published studies, we determined therole of these cytokines in cancer progression (SI Appendix, TableS2). Among the 26 affected cytokines, the macrophage colonystimulating factor (M-CSF) and granulocyte M-CSF (GM-CSF)play an essential role in the regulation of TAMs (39, 40). TAMspromote tumor progression and are resistant to various chemo-therapeutic agents (41, 42) in prostate cancer (43) by differentiatinginto either cytotoxic (M1) or tumor growth promoting (M2) states(44). Therefore, to study the implications of increased NO levels onTAMs, we evaluated the markers of proinflammatory (M1) andantiinflammatory (M2) macrophages in tumors from mice treatedwith GSNO versus PBS. We found that following therapy with NO,expression of M2 macrophage markers (F4/80, CD206, Arginase)was suppressed and expression of M1 macrophage marker (iNOS)was increased (Fig. 3 D–G). This indicates that TAMs, a significantcomponent of the antiinflammatory cell (M2) that infiltrates inprostate cancer (44), are suppressed by increased NO levels.

NO Influences TAMs by Targeting Their Activity and Differentiation.Recent studies have suggested the importance of phospho-ERK1/2 levels with respect to combinations of lactate and hyp-oxia that eventually affect the fate of TAMs (45–48). Therefore, toverify whether TAM activity is sensitive to increased levels of NO,we checked the levels of pERK in tumors from mice which receivedGSNO treatment and compared them with tumors from PBS-treated mice using immunostaining and Western blot. We found

Fig. 1. Increased levels of NO affect levels of testosterone (T), FSH, and LH.(A) To validate the impact of increased NO levels on T, FSH, and LH, C57/BL6mice were treated with 10 mg/kg GSNO for 7 d and levels of T, FSH, and LHwere checked. (B) Additionally, C57/BL6 mice were kept on the oral dosage ofGSNO at 10 mg per cage per week for 5 wk. Brains from i.p. GSNO-treatedmice were harvested and analyzed for the presence of GnRHR (C), showingthat NO levels affect major pathways of production of hormones (T, LH, FSH)by influencing hypothalamic-pituitary-gonadal axis regulation. (Scale bars: C,first three columns, 750 μm; C, Right, 100 μm.)

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that levels of pERK were suppressed upon GSNO treatment (Fig. 4A and B and SI Appendix, Fig. S5D). However, total levels of ERKprotein remained similar in all of the conditions (Fig. 4A), In ad-dition, we determined the impact of increased levels of NO on otherfactors which are markers of TAM activity, such as VEGF (an-giogenic marker), androgen receptor (AR), and androgen receptorsplice variant 7 (AR-V7) (a critical determinant of resistance de-velopment in CRPCs) (49, 50), and found their levels to be con-sistently suppressed upon treatment of tumors with GSNO (Fig. 4 AandC and SI Appendix, Fig. S5 A–C). Together, the results suggest astrong effect of NO levels on regulation of TAM activity.Another aspect of TAM regulation that we focused on is TAM

differentiation. Previous studies showed that tumor-derived factorslike IL-34 and M-CSF educate macrophages to become the alter-natively activated M2 type to promote angiogenesis, tissue remod-eling, and immune suppression (13, 24). For differentiation ofTAM, the binding of cytokines (IL34, CSF) to the CSF1 receptor(CSF1R) is critical (7). Enrichment analysis suggested the signifi-cance of IL-34 and CSF1R in resistance development in CRPCpatients (Fig. 4D and SI Appendix, Fig. S3 A–E). Therefore, weexplored the implications of increased NO levels on 22RV1 cells inthe presence or absence of GW2580 (CSF1R inhibitor) at varyingconcentrations (0, 0.5, 10, 25, and 50 μM, respectively). Upon thesuppression of IL-34–CSF1R interaction, expression of PSA andTMRPSS2 was abrogated (Fig. 4E). Taken together, decreasedlevels of pERK, AR in tumors, and abrogated levels of PSA andTMRPSS2 upon CSF1R inhibition strongly supports the regulatoryrole of NO on TAM activity and differentiation.

Short Half-Life of NO Does Not Affect Its Long-Term Impact on TAMs.We evaluated the efficacy of increased NO levels in the re-duction of CRPC tumor considering the short half-life of NO.For this, the CRPC cells (5 million cells per mouse) that wereisolated from animals in the two treatment groups (PBS andGSNO) were xenografted s.c. into castrated SCID mice (n = 4 pergroup) (secondary xenograft). Tumors were then allowed to grow

for a period of 4 wk (Fig. 5A). The results revealed that the overalltumor burden was significantly suppressed (Fig. 5B), with a re-duction in the number of Ki-67–positive cells in animals that re-ceived cells from GSNO-treated mice (SI Appendix, Fig. S4). Inaddition, cells staining positive for M2 macrophages markers (F4/80, CD206) were decreased in animals that received cells fromGSNO-treated mice (Fig. 5 C and D). Taken together, the effectof GSNO in secondary xenografts indicates the potential long-term therapeutic implications of increased NO levels on CRPC.

DiscussionOur study reveals an essential role for NO in CRPC tumorsuppression. We found that increased levels of NO, which areassociated with lowering LH and T under physiological condi-tions, lead to inhibition of prostate tumor growth in a cell non-autonomous manner. In addition, we established that increasedlevels of NO down-regulate the translational activity of AR,leading to a decreased expression of AR-V7 and pERK levels intumors. In addition, we showed that NO levels have the potentialto influence differentiation of TAM by affecting ligands like IL-34 and the receptors to which they bind (CSF1R). NO levelscontribute to a decrease in the M2 macrophages (F4/80, CD206)and the induction of M1 macrophages (iNOS). Finally, westudied whether the short half-life of NO affects its efficacy ontumor macrophages. The results show that tumor burden wassignificantly suppressed in mice that received cells from micetreated with GSNO. In addition, the M2 macrophage marker wasdecreased in the tumor grafts that acquired cells from GSNO-treated mice, thus supporting the potential long-term therapeu-tic implications of increased NO levels on CRPC.Several studies have demonstrated the tumoricidal effects of NO

in other cancer types. Schleiffer et al. (51) showed that NO likelyplays a role in neoplastic changes in a rat model of colon cancer.This work revealed that preneoplastic changes were promoted in thecolon by decreasing the release of NO through the inhibition of

Fig. 2. Increased NO levels suppress tumor burden in CRPC mouse model (castrated SCID mice with s.c. xenograft of 22RV1). (A) Experiment with in vivoxenograft. (B) Xenograft tumors isolated from both flanks after 2 wk of treatment (PBS/GSNO) (each cup represents tumors from one mouse). (C) Animalweight. (D) Tumor volume. (E) Tumor weight. GSNO-treated CRPC mouse models have fewer necrotic areas and fewer proliferating cells in tumor grafts.(F) Relative comparison of nitrate levels between control and GSNO-treated mice. (G) H&E staining, showing the sections from tumor xenografts that receivedPBS (control) vs. treatment with GSNO (10 mg/kg per d i.p.). Grafts from PBS-treated mice have more areas of necrosis. (H) Ki-67 immunostaining of sectionsshowing that proliferation is suppressed in GSNO-treated grafts. (Scale bars: 500 μm.)

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iNOS (51). A separate study (24) demonstrated the role of NOin renal cancer cells and the ability of NO to inhibit tumorgrowth, as authors induced NO production by transfection oftumor cells with iNOS. Their data indicate that the high levelsof NO are associated with inhibition of tumor growth and a

reduction in lung metastases (24). The tumoricidal effects ofNO are understudied in prostate cancer. Usually, tumor growthin therapeutics of prostate cancer is controlled through steroidmanagement by blocking the AR or by decreasing circulatingandrogens. However, this method has been limited due to the

Fig. 3. NO targets CRPC in cell nonautonomous manner. (A) Cell proliferation assay (MTT) using GSNO concentrations of 0, 5, 10, 25, 50, and 100 μM on22RV1 cells showed no specific inhibition, indicating effects of NO on 22Rv1 cells are likely cell nonautonomous. (B) Relative expression of PSA and TMRPSS2upon varying concentrations of GSNO ranging from 10, 25, 50, and 100 μM at RNA levels. (C) Twenty-six cytokines (CCL27, CD54, TIMP-1, ACRP30, G-CSF, AR,IL-17A, beta NGF, IL-2 R alpha, CCL28, Axl, CCL7, CCL17, CXCL6, IL-1 F2, M-CSF, TGFBeta 3, CXCL13, BMP-6, CCL23, NT-3, CCL11, CCL1, IL-5, IL-6, and IGFBP4)that were found to be suppressed more than 1.5-fold in tumors isolated from mice that received GSNO treatment compared with control mice that receivedPBS. (D) Impact of NO on TAM M1 macrophage (iNOS) induced and M2 (CD206 and Arginase-1) at RNA levels. (E–G) Impact of GSNO on M2 (F4/80 and CD206)and M1 macrophage (iNOS) at protein levels. (Scale bars: E, 750 μm; F, 100 μm; G, 75 μm.)

Fig. 4. Impact of NO on TAM activity and differentiation. To validate the impact of increased levels of NO on TAM activity, the expression of pERK, ERK, AR-V7, and VEGF was checked in tumor grafts isolated from animals treated with PBS or GSNO. (A) Western blot confirmed reduced levels of AR-V7 as well aspERK upon increased NO. Immunostaining confirmed a reduced number of cells staining positive for pERK (B) and VEGF (C) upon increased NO levels. (Scalebars: B, 500 μm; C, 100 μm.) (D) To validate the impact of increased levels of NO on TAM differentiation, we evaluated expression of IL-34 in 22RV1 cellstreated with 10, 25, 50, and 100 μMGSNO. (E) To establish the significance of suppression of IL-34–CSF1R interaction, CSF1R was blocked by GW2580 at 0.5, 10,25, and 50 μM, followed by treating 22RV1 cells with GSNO. Inhibiting CSF1R abrogated GSNO suppressive impacts on PSA and TMPRSS2 levels.

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progression of the disease to a castration-resistant prostate cancerin which steroid manipulation becomes ineffective (52). Previousstudies have demonstrated that even modest increases in AR andARV7 expression may contribute to the development of resistanceand progression of disease (14702632) (24909511). Therefore,increased NO capability to decrease AR expression (SI Appendix,Fig. S5) may promote the susceptibility of PCa tumor cells tocurrent therapies. Recently, it has been reported that elevated NOlevels are able to inhibit tumor growth in androgen-dependent aswell as CRPC cell lines such as LNCAP (30). However, the in vivoregulatory mechanisms behind the impact of increased NO levelson CRPC tumors have largely been unknown. Our study reveals thatthe TME could be the target of NO for suppression of tumori-genesis in CRPC cell lines like 22RV1.There are several strengths and limitations in our study. The study

reports four major findings: (i) This investigation demonstrates thatNO levels increased by GSNO are capable of arresting tumor bur-den in highly tumorigenic 22RV1 CRPC xenografts; (ii) the workshows that NO deffects on CRPC are cell nonautonomous effectsand are targeting the TME; (iii) increased NO affects both activityand differentiation of TAMs in CRPC; and (iv) the study validatesthe efficacy and durability of NO using a secondary xenograftmouse model. The limitations of our study are as follows: (i) Theexact mechanisms by which NO-affected TAM differentiation re-main unexplored; (ii) increased NO levels are capable of affectinghypogonadism and lead to CRPC tumor suppression, thereforeleading to the strong possibility that the tumors inhibition could be

profound in noncastrate mice. However, this assumption still needsto be explored; (iii) the dose-dependent effect of NO on CRPC isnot validated; and (iv) we have used 22Rv1 cells as a model cell lineto investigate the implications of NO; however, use of cells fromhuman xenograft models of CRPC which were beyond the scope ofthe present study, but are ongoing in our laboratory, may furtherconfirm and extend the findings discovered in this current study.In conclusion, this study shows the regulatory role of NO in the

TME of CRPC. To better understand the role of NO in the ther-apeutics of CRPC, a further in-depth evaluation of other NO do-nors and their effects on the TME of CRPC as well as a long-termfollow-up to determine how the loss of function of NO could revertthe TAMs and affect CRPC is currently being addressed.

Materials and MethodsAnimal experiments were carried out in compliance with the InstitutionalAnimal Care and Use Committee of University of Miami. Molecular analyseswere performed using standard procedures. A more detailed description andadditional data are provided in SI Appendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank all the mentors (Dr. Dipen J. Parekh),collaborators (Dr. Alan Pollack, Dr. Chad Ritch), and interns (AysswaryaManoharan, Khushi Shah) for their insights, suggestions, and support duringthis study. Additionally, we thank the American Urological AssociationResearch Scholar Award and Stanley Glaser Award (for R.R.) and the SexualMedicine Society of North America (SMSNA) (for H.A.). J.M.H. is supported byNIH Grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387,and 5UM1 HL113460, and the Soffer Family Foundation.

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Fig. 5. Long-term implications of NO on CRPC. (A) To validate the efficacy of NO on CRPC, we used secondary xenograft models in which castrated SCID micewere xenografted s.c. with 22RV1 cells followed by GSNO treatment in half of them for 4 wk. Cells from the two groups (control and experimental) of micewere harvested and reinjected into another set of castrated SCID mice, which were maintained for 4 wk but not treated (secondary xenograft). (B) The lattergroup showed a significant decrease in tumor burden. Mice that received cells from GSNO-treated animals showed a lower percentage of cells stainingpositive for M2 macrophages like F4/80 (C) and CD206 (D). (Scale bars: 100 μm.)

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