a jak2-selective inhibitor potently reverses the immune...

Biochemical Pharmacology xxx (2017) xxx–xxx

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Biochemical Pharmacology

journal homepage: www.elsevier .com/locate /b iochempharm

A Jak2-selective inhibitor potently reverses the immune suppression bymodulating the tumor microenvironment for cancer immunotherapy

http://dx.doi.org/10.1016/j.bcp.2017.08.0190006-2952/� 2017 Elsevier Inc. All rights reserved.

⇑ Corresponding authors.E-mail addresses: [email protected] (J. Han), [email protected] (L. Dong).

1 These authors contributed equally to this work.

Please cite this article in press as: W. He et al., A Jak2-selective inhibitor potently reverses the immune suppression by modulating the tumor mvironment for cancer immunotherapy, Biochem. Pharmacol. (2017), http://dx.doi.org/10.1016/j.bcp.2017.08.019

Wei He a,1, Yi Zhu b,1, Ruoyu Mu a, Jinzhi Xu a, Xiaoyi Zhang c, Chunming Wang e, Qiu Li e, Zhen Huang a,Junfeng Zhang a,d, Yi Pan b, Jianlin Han b,⇑, Lei Dong a,⇑a State Key Laboratory of Pharmaceutical Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue,Nanjing 210093, Chinab School of Chemistry and Chemical Engineering, State Key laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, ChinacDepartment of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USAd Jiangsu Provincial Laboratory for Nano-Technology, Nanjing University, Nanjing, Chinae State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 June 2017Accepted 25 August 2017Available online xxxx

Chemical compounds studied in this article:DMSO (PubChem CID: 679)DAPI (PubChem CID: 2954)PMA (PubChem CID: 27924)Lipopolysaccharides (PubChem CID:53481793)Sodium citrate anticoagulant (PubChemCID: 71474)Phenylmethylsulfonyl Fluoride (PubChemCID: 4784)MgCl2 (PubChem CID: 5360315)Sodium Orthovanadate (PubChem CID:61671)ATP (PubChem CID: 5957)Tris (PubChem CID: 6503)

Keywords:Tumor associated macrophagesRegulatory T cellsJak2-STAT3 pathwayTumor immunotherapy

Small molecule therapeutics can be potent tools for cancer immunotherapy. They may be devised to tar-get the tumor associated macrophages (TAMs) and regulatory T cells (Treg), which are major immuno-suppressive cells in the tumor microenvironment. The infiltration and functionalization of these cells,which essentially promote tumor development, are mediated by the hyper-activation of the Jak-STAT3signaling pathway. Here, we demonstrated that compound 9#, a novel inhibitor of Jak2, could suppressJak2-STAT3 signaling in macrophages (peritoneal macrophages and THP-1 cells) and direct the macro-phages toward the pro-inflammatory (M1-like) phenotype. When tested in ex vivo TAM culture andin vivo tumor models, compound 9# could reverse the phenotype of TAM from M2- to M1-type by pro-moting IL-12 expression. Further study suggested that compound 9# also inhibited the induction of Tregboth in vitro and in vivo via blockage of Jak2 signaling. Finally, compound 9# potently increased the fre-quency and anti-tumor activity of CD4+ and CD8+ T lymphocytes, leading to effective suppression oftumor growth. Taken together, our findings indicated that compound 9# could be a potential candidateof small molecule therapeutics for cancer immunotherapy.

� 2017 Elsevier Inc. All rights reserved.

1. Introduction

Therapeutic approaches targeting the immune system holdgreat promise for the treatment of cancers [1,2]. Current majorstrategies for cancer immunotherapy rely on the use of proteindrugs or cell-based therapies, which both face the challenges of

instability, technical complexity and high cost. In comparison,small molecule chemicals that are stable and effective in modulat-ing the immune system may be desirable alternatives [3]. Usingsmall molecule drugs to target and modulate tumor immunityhas an obvious advantage – the tumor cells (malignant cells) easilyacquire resistance to the therapeutic agents due to their geneticinstability [4], but the immune cells are relatively more stableand less likely to develop such resistance [5]. Therefore, designingand screening small molecule inhibitors of the immune signalingthat regulates tumor development may be of great potential.

icroen-

http://dx.doi.org/10.1016/j.bcp.2017.08.019

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2 W. He et al. / Biochemical Pharmacology xxx (2017) xxx–xxx

Tumor-associated macrophages (TAMs) and regulatory T cells(Treg) are among the main types of immunosuppressive cells intumor microenvironment. They are the key players in organizingthe immune escape of tumors, which has become a main barrierfor cancer immunotherapy [6,7]. Clinical investigations demon-strated the relevance between high TAMs/Treg invasion and thepoor prognosis of cancer patients, and recent studies suggestedthat re-educating TAMs into M1 type or locally eliminating Tregwere both effective in treating cancers [8–12]. Therefore, newsmall molecule agents capable of modulating TAM phenotype orinhibiting Treg generation may be promising tools of cancerimmunotherapy.

The Janus kinase and signal transducer and activator of tran-scription 3 (Jak-STAT3) signaling are constitutively activated inimmune cells in the tumor environment, including TAMs and Treg[13–15]. Recent studies showed that activation of Jak-STAT3 notonly contributed to the polarization of TAMs and the differentia-tion of Treg, but also regulated the functions of these cells, espe-cially those in the process of cancer immune escape [16,17]. Also,Jak-STAT3 pathway is activated in the majority of cancer cells[18,19]. In addition, activation of Jak-STAT3 pathway could alsopromote cancer cells and immune cells to secrete many immuno-suppressive cytokines such as IL-10 and IL-6. Thus, this pathwaycould play a role in mediating crosstalk between tumor cells andtheir immunological microenvironment [16]. Therefore, thehyper-activation of Jak-STAT3 pathway may promote tumor pro-gression through multiple mechanisms and inhibition of its activa-tion may inspire a new strategy for cancer therapy.

In this study, by screening a group of fluorinated b-amino-ketone molecules, we found that compound 9# could selectivelyinhibit the activity of Jak2 kinase. Compound 9# was further testedfor its effect on the phenotype of TAMs and induction of Treg in aseries of in vitro and in vivo studies, and evaluated for its anti-tumor activity in different tumor-bearing murine models. Ourresults suggested that compound 9# could become a promisinglead candidate for further development of Jak2 inhibitors for can-cer immunotherapy.

2. Materials and methods

2.1. Reagents

Fluorinated b-amino-ketones (90% or higher purity) were syn-thesized according to the previous reported method [20], whichwere dissolved in dimethylsulfoxide (DMSO) as a 80 mM shocksolution and stored at 4 �C. DMSO, Lipopolysaccharide (LPS,Escherichia coli, L2630, Sertype O111:B4), Phorbol 12-myristate13-acetate (PMA), Phenylmethylsulfonyl Fluoride, Sodium Ortho-vanadate and ATP were purchased from Sigma-Aldrich (St. Louis,Missouri, USA). DMEM, RPMI 1640, penicillin/streptomycin antibi-otic mixture and fetal bovine serum (FBS) were purchased fromLife Technologies (Grand Island, New York, USA). Other chemicalreagents were ordered from Sangon Biotech (Shanghai, China).

2.2. Cell lines

B16F10 cells were propagated in DMEM supplemented with10% heat-inactivated FBS, 100 U/ml penicillin, and 0.1 mg/mlstreptomycin. 4T1 cells and THP-1 cells were maintained inRPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. All cell lines were pur-chased from ATCC (B16F10 cells, ATCC� CRL-6475TM; 4T1, ATCC�

CRL-2539TM; THP-1 cells, ATCC� TIB-202TM) and maintained accord-ing to their recommendations.

Please cite this article in press as: W. He et al., A Jak2-selective inhibitor potenvironment for cancer immunotherapy, Biochem. Pharmacol. (2017), http://dx.d

2.3. Mice

BALB/c mice, and C57BL/6J mice (all female, 6–8 weeks old)were purchased from the Animal Centre of Yangzhou University(Yangzhou, China). Animals were acclimatized in a ventilated,temperature-controlled room (23 �C) with a 12 h light/12 h darkcycle. All animals had free access to rodent chow and water andwere treated in strict accordance with the Nanjing Universityguidelines (Permit NO. 2011-039).

2.4. Tumor models

Tumor studies were performed as previously described [21]. Togenerate the heterotopic tumor model, 1 � 106 cells were injectedsubcutaneously into the left armpit of the animals. Tumor-bearingmice were randomized based on tumor volume prior to the initia-tion of treatment, which was initiated when average tumor volumewas at least 50 mm. Compound 9# was given intratumoral injec-tion, as indicated in sterile PBS. We measured the tumor size withcaliper and weighted the tumor samples upon harvest. In someexperiments, tumor cells were mixed with TAMs (2.5:1) treatedwith or without compound 9# and then injected into mice. Tumordiameters were measured every fourth day, and we weighted thetumor samples upon harvest.

2.5. Experimental protocol of docking study

Molecular docking of the compound 9# binding the three-dimensional X-ray structure of Jak family (Jak1, PDB code: 4I5C;Jak2, PDB code: 3E62; Jak3, PDB code: 5TOZ) was carried out usingDiscovery Studio (version 3.5) as implemented through the graph-ical user interface DS-CDOCKER protocol. The aforementionedcompound 9# was constructed, minimized and prepared. The crys-tal structures of the protein complex were retrieved from the RCSBProtein Data Bank (http://www.rcsb.org/pdb/home/home.do). Allbound waters and ligands were eliminated from the protein.Molecular docking was performed by inserting molecules intothe binding pocket of Jak family (Jak1, 2, 3) based on the bindingmode. The types of interactions between the docked proteins withligand-based pharmacophore model were analyzed after the end ofmolecular docking.

2.6. Mouse peritoneal macrophages and TAMs isolation and treatment

Mouse peritoneal macrophages were isolated and culturedaccording to a published method [22]. Purified macrophages (pur-ity > 90%) were incubated in 6-well plates in RPMI-1640 mediumwith 10% (V/V) FBS at 37 �C in a humidified atmosphere of 5% CO2.

TAMs from tumor tissues were isolated according to a pub-lished method [23] and cultured in RPMI-1640 medium containing10% FBS. The purity and viability of TAMs were more than 90%,which were verified by flow cytometry (F4/80) and trypan bluestaining, respectively.

2.7. Cell viability assay

Mouse peritoneal macrophages (1 � 105/well), THP-1 cells(1 � 105/well) and cancer cell lines (5 � 103/well) were culturedwith various concentrations of compound 9# (2.5–80 lM) for3 days in 96-well plates. After culture, cell viability was deter-mined by CCK-8 assay.

tly reverses the immune suppression by modulating the tumor microen-oi.org/10.1016/j.bcp.2017.08.019

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W. He et al. / Biochemical Pharmacology xxx (2017) xxx–xxx 3

2.8. Cell-free kinase activity assays

IC50 values for compound 9# were determined using aluminescence-based kinase assay with recombinant Jak1, Jak2and Jak3 proteins, as described previously [24].

2.9. Jak2 kinase activity assay

Jak2 was immunoprecipitated from mouse peritoneal macro-phages and THP-1 cell lysate using 80 ll rabbit anti-jak2 antibod-ies and 300 ll protein A/G agarose slurry. The total antibody-Jak2protein complex was resuspended in 300 ll of 50 mM Tris, pH7.4 containing 5 mM MgCl2, 0.1 mM sodium orthovanadate, andwas divided into 10 equal parts; 30 ll aliquots were then incu-bated with compound 9# (2.5–40 lM) or buffer alone on ice for20 min. Kinase reactions were initiated with the addition of 10 llATP (a final concentration of 3 mM in reaction buffer) and incu-bated at 25 �C for 45 min before quenching with the addition of5�-Laemmle sample buffer . Samples were heated to 100 �C for10 min, centrifuged at 12,000g for 10 min, and the supernatantfraction was resolved by SDS-PAGE, and then transferred to nitro-cellulose membranes. After blocking (1.5 h at room temperature)with 5% nonfat milk in PBS-0.1% Tween-20, membranes were incu-bated overnight with phosphor-Jak2, Total-Jak2 antibodies (Cellsignaling technology, Danvers, MA, USA) at 4 �C, and then exposedto secondary antibody. Immunocomplexes were visualized usingenhanced chemiluminescence detection system (ECL). Thephosphor-Jak2 level was regard as a measure of its kinase activity.We quantify Jak2 protein via controlling the same volume ofantibody-Jak2 protein complex, and 30 ll aliquots were used totest Jak2 kinase activity assay in vitro. In addition, western blottingdemonstrated the level of the total-Jak2 proteins in each groupwasequal.

2.10. Primary CD4+ T-cell purification and the in vitro differentiationinto Treg

CD4+ T cells were purified using BD IMagTM Anti-Mouse CD4Magnetic Particles according to the manufacturer’s instructions(BD Biosciences, San Jose, CA, USA). We confirmed the purity ofCD4+ T cells was more than 90% with flow cytometry. For in vitroTreg differentiation, sorted cells were activated with 2 lg/mlanti-CD3 and anti-CD28 (eBioscience, San Diego, CA, USA) and theninduced to differentiate into Treg by the supplementation of 5 ng/ml TGF-b1 (Pepro Tech, Rocky Hill, NJ, USA) and 5 lg/ml anti-IFN-cantibodies (biolegend, San Diego, CA, USA).

2.11. RNA interference

Mouse Jak2 siRNA (sense: 50-GUCCACCCGUGGAAUUUAUTT-30

and antisense: 50-AUAAAUUCCACGGGUGGACTT-30), and humanJak2 siRNA pool (a, 50-CGAAUAAGGUACAGAUUUC-30; b, 50-UUACAGAGGCCUACUCAUA-30; c, 50-AAUCAAACCUUCUAGUCUU-30; and d,50-GGAAUGGCCUGCCUUACGA-30) were purchased from Gene-Pharm (Shanghai, China). 100 nM siRNA were transferred to a 4-mm electrode gap cuvette (Molecular Bioproducts, Germany) andeither Opti-MEM was filled up to a total volume of 50 ll. 50 ll ofthe cell suspension (containing 2 � 105 cells) in the Opti-MEMwere added to the cuvette and mixed by pipetted to give a finalvolume of 100 ll. The cuvette was immediately pulsed at a rangeof voltage and capacitance levels (400 V, 150 lF, and 100X) usinga Gene Pulser electroporation (Bio-Rad, Hercules, USA). The elec-troporated cells were incubated at 37 �C for 10 min before beingtransferred into 6-well plates. The treatments were performed24 h after the siRNA transfection.


2.12. Flow cytometry analysis

To prepare single-cell suspensions for flow cytometry, spleenand tumor tissues were dissected into fragments and gently disso-ciated under 70 lm cell strainers for single-cell isolation, andmouse peripheral blood samples were collected at 1.5 ml tube con-taining 3.8% sodium citrate anticoagulant. After red blood cell lysis,single-cell suspensions were washed, and resuspended in 1� PBSwithout Ca and Mg. 5 � 105 cells were blocked in 100 ll of 1%BSA at 4 �C for 30 min. For the analysis of CD4+ T cells and CD8+

T cells, cells were surface labelled with fluorescently conjugatedantibodies against CD4 or CD8. For analysis of Treg, cells werestained for surface markers (CD4 and CD25), fixed and made per-meable with Foxp3 Staining Buffer, and stained with fluorescencelabelled antibodies (Foxp3). Samples were run with FACSCalibur(BD Bioscience, San Jose, CA, USA) and analyzed using FLOWJOsoftware (Treestar Inc, Ashland, USA). Antibodies against CD4,CD8, CD25 and Foxp3 were obtained from biolegend (San Diego,CA, USA).

2.13. Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from cells using TRIzol reagent (Invitro-gen, Carlsbad, CA, USA) following the manufacturer’s instruction.The quality and quantity of total RNA were verified by an Eppen-dorf Biophotometer Plus (Eppendorf AG, Hamburg, Germany).Real-time qPCR was performed using LightCycler FastStar DNAMaster SYBR Green I (Roche Diagnostics) according to the manu-facturer’s protocol. Primers for b-actin were used as internal con-trols. Mouse NOS2 sense: 50-CCAAGCCCTCACCTACTTCC-30; MouseNOS2 antisense: 50-CTCTGAGGGCTGACACAAGG-30; Mouse MHCIIsense: 50-CATCTGCTCACGAGGTCTGGA-30; Mouse MHCII antisense:50-TGGCACTGGAGTGGCAAATAG-30; Mouse Arg1 sense: 50-CTCCAAGCCAAAGTCCTTAGAG-30; Mouse Arg1 antisense: 50-AGGAGCTGTCATTAGGGACATC-30; Mouse Ym1 sense: 50-AGAAGGGAGTTTCAAACCTGGT-30; Mouse Ym1 antisense: 50-GTCTTGCTCATGTGTGTAAGTGA-30; Human NOS2 sense: 50-TTGCTGTGCTCCATAGTTTCCA-30; Human NOS2 antisense: 50-GCGATTTCTTCAGTTTCTCTCCAT-30; Human CCL18 sense: 50-CTCTGCTGCCTCGTCTATACCT-30; Human CCL18 antisense: 50-CTTGGTTAGGAGGATGACACCT-30; b-actin sense: 50-GGTGTGATGGTGGGAATGGG-30; b-actin antisense: 50-ACGGTTGGCCTTAGGGTTCAG-30.

2.14. Histological and immunofluorescence analysis

Tumor samples were fixed in Bouin’s buffer, embedded in paraf-fin and sectioned. For histologic analyses, the slices were counter-stained with haematoxylin and eosin (H&E). Forimmunofluorescence analysis, the slices were washed with PBSand blocked with 5% BSA for 1 hour, and incubated with the follow-ing primary antibodies: rabbit anti-mouse interleukin-10 (IL-10),rabbit anti-mouse interleukin-12 (IL-12), rabbit anti-mouse VEGF,rabbit anti-mouse MMP-9, rabbit anti-mouse platelet endothelialadhesion molecule (CD31), and hamster anti-mouse interferon-gamma (IFN-c) at 4 �C overnight. The sections were washed withTris-buffered saline three times and subsequently stained withthe following secondary antibodies: Alexa 488 labelled donkeyanti-hamster, Alexa 546 labelled donkey anti-rabbit at room tem-perature for 60 min and washed as above. At last, the sections werestained with the nuclei being counterstained with 406-diamidino-2-phenylindole (DAPI) at room temperature for 5 min. Sampleswas imaged using a Nikon confocal microscope. Each stainingwas performed with three parallel samples and samples stainedwith secondary antibodies alone were set to determine the back-ground threshold. Primary antibodies against IL-10, IL-12, VEGFand MMP-9 were purchased from 4A Biotech Co., Ltd (Beijing,




China). Antibodies against IFN-c were obtained from biolegend(San Diego, CA, USA). Antibodies against CD31 were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Alexa 488 labelleddonkey anti-hamster, Alexa 546 labelled donkey anti-rabbit wereobtained from Life technologies (Carlsbad, CA, USA).

2.15. Western blotting

For western blotting, cellular protein extract were prepared bylysing cells in RIPA buffer containing 1 mM PhenylmethylsulfonylFluoride (PMSF), and the protein concentration was determinedusing BCA protein assay kit (Beyotime Biotechnology, Shanghai,China). 50–100 lg of protein was subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to nitro-cellulose membranes. After blocking (1.5 h at room temperature)with 5% nonfat milk in PBS-0.1% Tween-20, membranes were incu-bated overnight with indicated antibodies at 4 �C, and thenexposed to secondary antibody. Immunocomplexes were visual-ized using enhanced chemiluminescence detection system (ECL).Antibodies recognizing phosphor-Jak2, Total-Jak2, phosphor-STAT3, total-STAT3 and GAPDH were obtained from cell signalingtechnology (Danvers, MA, USA).

2.16. ELISA analysis

The concentrations of IL-10, IL-12, VEGF, MMP-9, TNF-a, IL-12p40 and IFN-c in cell-culture supernatant or tumor tissues werequantified by using commercially available ELISA Kits (4A BiotechCo., Ltd, Beijing, China). Assays were performed according to themanufacturer’s protocol and read at 450 nm by using a microplatereader (Thermo scientific).

2.17. Data analysis

Results are expressed as the means ± standard error (mean-s ± SEM) of at least 3 independent experiments, and each dosageor treatment was tested in triplicate. Differences between groupswere compared using the Mann-Whitney U test and, if appropriate,by a one-way ANOVA test. Multiple comparisons between thegroups were performed using the S-N-K method. NS indicatednot significant statistically. The difference was considered signifi-cant when p � 0.05.

3. Results

3.1. Compound 9# is a selective Jak2 kinase inhibitor and inhibitsJak2-STAT3 pathway in vitro

Sixteen fluorinated b-amino-ketones against Jak2 kinase wereevaluated by in vitro Jak2 kinase assays. Out of the sixteen com-pounds, compound 9# was found to have highest inhibitory activa-tion for Jak2, and was chosen for further characterization (Table 1).The molecular structure of compound 9# is shown in Fig. 1A,which was prepared from pentafluoro-b-di-ketone hydrates and(S)-N-tert-butanesulfinyl (3,3,3)-trifluoroacetaldimine. Due to thehigh homology in the adenosine triphosphate (ATP) binding pocketamong the members (Jak1, 2, 3) of Jak family, and the specificity ofcompound 9# to inhibit the Jak family kinases’ activity was exam-ined against purified Jak1, Jak2 and Jak3 proteins. Compound 9#inhibited all three Jak kinases at different IC50 value(2.15 � 15.36 lM) in in vitro kinase assays (Fig. 1B). Jak2 was themost sensitive one to compound 9#, indicating relatively signifi-cant selectivity and specificity for Jak2 comparing to Jak1 andJak3 in this assay.


Docking study was also performed to predict the binding affini-ties of compound 9# with Jak family (Jak1, 2, 3), and the generalworkflow was described in the method. After the computationalcalculation, the best poses of compound 9# docking with the bind-ing sites of Jak family (Jak1, 2, 3) were validated with CDOCKER-interaction energy values of �39.29, �41.14, and �33.95 kcal/mol,respectively (Table 2). From the results, compound 9# bindingwith Jak2 displayed the lowest CDOCKER interaction energy, whichsuggested compound 9# could bind with Jak2 more stably, com-pared with the bindings with Jak1 and Jak3. Furthermore, the bind-ing model of compound 9# with Jak2 was analyzed. The dockingresults (Fig. 1C and D) revealed that seven amino acids Lys-857,Arg-980, LEU-855, Val-863, Ala-880, Asn-981 and Leu-983 locatedin the binding pocket of Jak2 protein played major roles in interact-ing with compound 9# by forming two hydrogen bonds, three car-bon hydrogen bonds, four halogen bonds, and four Pi-alkyl bonds.The molecular docking results, along with the biological assay data,suggested that compound 9# was a potential inhibitor of Jak2.

Autophosphorylation of Tyr 1007 is required for high catalyticactivity of Jak2, and blockade of autophosphorylation of Jak2 atTyr 1007 suppresses tyrosyl phosphorylation of downstream STATpathway. We found that compound 9# could inhibit Jak2autophosphorylation in a dose-dependent manner (Fig. 1E). STAT3is one direct target of the phosphorylation of Jak2. We investigatedwhether compound 9# decreased STAT3 phosphorylation inmacrophages. Results in Fig. 1F demonstrated that together withphosphor-Jak2 (P-Jak2), the level of phosphor-STAT3 (P-STAT3) inperitoneal macrophages and THP-1 cells was significantly sup-pressed by compound 9# in a dose-dependent manner. Compound9# at 40 lM could completely inhibit the phosphorylation ofSTAT3 in both cells. Additionally, compound 9# showed little cyto-toxicity to peritoneal macrophages and THP-1 cells at the concen-trations of 2.5–80 lM (Fig. 1G), which excluded the possibleinfluence from the cytotoxicity to the STAT3 phosphorylation. Col-lectively, these results provide evidence that compound 9# couldefficiently inhibit the phosphorylation of Jak2 and STAT3 in macro-phages with prominent specificity.

3.2. Compound 9# promotes macrophages toward M1 phenotypepolarization via inhibiting Jak2-STAT3 pathway

Jak2-STAT3 signaling is key for macrophage polarization. Highlevel p-STAT3 was found in M2 macrophages or TAMs. BlockingJak2-STAT3 pathway could result in increasing amounts of pro-inflammatory mediators, including cytokines and nitric oxide, afterLPS stimulation [25,26]. We detected the effect of compound 9# onthe cellular phenotype in both peritoneal macrophages and THP-1cells. In peritoneal macrophages, compound 9# significantlyincreased NOS2, TNF-a and IL-12 expression and markedlyreduced Arg1 and IL-10 production in a dose-dependent mannerunder LPS stimulation (Fig. 2A and B). Similarly, in THP-1 cells,combination with PMA, compound 9# at different doses signifi-cantly increased the secretion of TNF-a and IL-12p40, enhancedthe NOS2 mRNA level (Fig. 2C), suppressed the IL-10 productionand decreased the CCL-18 mRNA level (Fig. 2D). In addition, wenoticed that compound 9# alone without the stimulation fromLPS or PMA could not induce significant change in those cytokinesand M1/M2-specific genes expression (Fig. 2A–D). Next, we usedJak2-specific siRNA assays to further confirm that whether Jak2-STAT3 signaling pathway by compound 9# mediates macrophagephenotype. Data show that no significant change in the level ofcytokines (IL-10, IL-12/IL-12p40 and TNF-a) and related genesmRNA expression (NOS2 and Arg1/CCL18) was observed in Jak2siRNA + compound 9# group with LPS/PMA stimulation, comparedwith Jak2 siRNA + LPS/PMA group (Fig. 2E–H). Analogously, theresults of western blot revealed that no difference in the level of



Table 1The chemical structures and Jak2 kinase IC50 values of the fluorinated b-amino-ketones.

Compd R Jak2 inhibition (IC50 lM)a Compd R Jak2 inhibition (IC50 lM)a

1 # 10.78 ± 2.38 9 # 2.35 ± 0.29

2 # 19.23 ± 3.01 10 # 6.33 ± 0.45

3 # 7.89 ± 1.13 11 # 9.67 ± 1.29

4 # 6.14 ± 0.91 12 # 11.22 ±1.66

5 # 11.35 ± 1.52 13 # 9.09 ± 1.76

6 # 7.79 ± 0.79 14 # 7.72 ± 1.38

7 # 9.98 ± 1.25 15 # 16.14 ± 2.69

8 # 6.62 ± 0.54 16 # 10.15 ± 2.35

a IC50 values were calculated from 2 to 3 determinations.


STAT3 phosphorylation was observed between these two groups(Fig. 2I).

TAMs are typical M2-like macrophages and play a key role incancer immune escape. We further analyzed whether compound9# could alter the TAMs’ phenotype and functions in vitro. Asshown in Fig. 3A and B, compound 9# significantly promoted theexpression of IL-12 and down regulated the IL-10 production inTAMs. Meanwhile, the increased M1 marker (NOS2 and MHCII)and decreased M2 marker (Arg1 and Ym1) were also observed.These results clearly demonstrated a remarkable functional changein TAM treated by compound 9#. Further data suggested that com-pound 9# reprogrammed TAMs via the Jak2-STAT3 signaling astreatment of TAMs with compound 9# could decrease the phos-phorylation level of Jak2 and STAT3 (Fig. 3C). Additionally, Silenc-ing Jak2 significantly shortened the effects of compound 9# onTAMs phenotype switch. No significant differences in relatedmacrophage markers expression and the level of P-STAT3 wereobserved between Jak2 siRNA group and Jak2 siRNA + compound9# group (Fig. 3D–F). Together, these results suggested that com-pound 9# could promote M1 phenotypic differentiation in macro-phages via inhibiting Jak2 – STAT3 signaling in vitro.

3.3. Compound 9# inhibited Treg differentiation via inhibiting Jak2activation in vitro

The Jak-STAT3 signaling is also key to the differentiation andfunctionalization of Treg. STAT3 neutralization could decreaseFOXP3 expression and prevented the acquisition of suppressivefunction [27]. Therefore, we examined whether compound 9#


could interfere the generation of immune suppressive Foxp 3+ Treg.By using a conventional in vitro Treg differentiation assay, wefound that compound 9# could significantly inhibit the differenti-ation of Foxp 3+ Treg from the purified naive CD4+ T cells incubatedwith CD3 and CD28 antibodies and TGF-b1. This effect was dosedependent and the generation of Foxp3+ Treg was almost com-pletely blocked by 40 lM compound 9# (Fig. 4A and B). IL-10and TGF-b contribute to the main immunosuppressive capabilityof Treg. Our further data in Fig. 4C demonstrated that the expres-sion of these two cytokines was gradually reduced following theincrease in the concentration of compound 9# in medium, indicat-ing a functional blockade in Treg. Furthermore, consistent withthat in macrophages, we found that compound 9# could alsodose-dependently reduce the phosphorylated Jak2 and STAT3 pro-tein level (Fig. 4D). Additionally, knocking down Jak2 significantlyweakened the effects of compound 9# on Treg differentiation(Fig. 4E and F) and function of expressing IL-10 and TGF-b (Fig. 4G),suggesting the direct involvement of Jak2 in compound 9# activity.Accordingly, the suppression of STAT3 phosphorylation by com-pound 9# was also abolished in Jak2 depleted Treg (Fig. 4H). Takentogether, the above data demonstrated that compound 9# had thesame activity in T cells as in macrophages to block the Jak2/STAT3signaling and suppress the generation of functional Treg.

3.4. The anti-tumor activity of compound 9#

TAMs and Treg are critical immunosuppressive cells in tumormicroenvironment and main contributors to the cells-mediatedimmunosuppression in tumor immune escape process. Therefore,



Fig. 1. Molecular structure of compound 9# and its selective inhibition of Jak2 kinase. A: Chemical structure of compound 9#. B: Compound 9# inhibitory activity (IC50)against selected kinases in the in vitro kinase assay. C and D: Molecular model depicting docking of compound 9# in the Jak2 ATP pocket. Key inhibitor-protein interactions,including two hydrogen bonds, three carbon hydrogen bonds, four halogen bonds, and four Pi-alkyl bonds are shown. E (1, 2) (a, b): Purified mouse and human Jak2 wereincubated in the presence of the indicated concentrations of compound 9# in kinase buffer, and autophosphorylation of Jak2 (as a measure of its enzymatic activity) weremeasured by Western blot. F (1, 2) (a, b): Macrophages were pre-incubated with different doses of compound 9# for 1 h and then cultured with lipopolysaccharide (LPS)(200 ng/ml) or phorbol-12-myristate-13-acetate (PMA) (50 ng/ml) for 30 min. The expressions of phosphorylated Jak2 (P-Jak2), total Jak2 (T-Jak2), phosphorylated STAT3 (P-STAT3) and total STAT3 (T-STAT3) in peritoneal macrophages and THP-1 cells were evaluated by Western blot. a, image of western blot; b, grey level of western blot. G: Theviability of peritoneal macrophages and THP-1 cells with different concentrations of compound 9# for 72 h. The data were normalized to DMSO treated cells. The results arerepresentative of three independent experiments and expressed as the means ± SEM. *P < 0.05, and **P < 0.01 compared with the ATP + 9# (without) group in Fig. 1E (1, 2) b.*P < 0.05, and **P < 0.01 compared with the LPS + 9# (without) group in Fig. 1F1b. *P < 0.05, and **P < 0.01 compared with the PMA + 9# (without) group in Fig. 1F2b.

Table 2The CDOCKER-interaction energy values when the best poses of compound 9#docking with the binding sites of Jak family.

Jak2 Jak1 Jak3

CDOCKER interaction energy (kcal/mol) �41.14 �39.29 �33.95


we investigated if compound 9# could prevent the immunosup-pressive effects of tumor and evoke the anti-tumor activity of theimmune system in two allograft mouse models implanted withthe murine breast cancer cell line, 4T1 and the murine melanomacell line, B16F10. As evidenced by the data in Fig. 5A–C, intratu-moral injection of compound 9# at different doses (20 and40 mg/kg) could significantly reduce the tumor weight and size,and prolong the mice survival period. In addition, all the tumor-bearing mice treated with compound 9# at dosage 40 mg/kg sur-vived through the test period (Fig. 5C). Fig. 5D shows the imageof tumor harvested after 14 days of different treatments. Fromthe results of H&E stained tumor sections in Fig. 5E, compound9# could promote tumor tissues necrosis in a dose-dependentmanner. To further support the antitumor efficacy of compound9# could be mediated via its effect on the TAM. We mixed tumorcells with TAM treated with or without compound 9# and then


injected into mice. The results demonstrated that mice injectedwith tumor cells plus compound 9#-treated TAMs could reducethe tumor size and weight, and retard tumor growth, comparedwith tumors in mice injected with tumor cells and TAMs(Fig. 5G–I). Additional analysis of the angiogenesis found that com-pound 9# treatment significantly reduced the concentration ofMMP-9 and VEGF in tumor tissues (Fig. 6A), and immunofluores-cence results also observed a decrease in the expression of thesecytokines in the tumor tissues (Fig. 6B and C). More importantly,CD31 staining-an endothelial marker-clearly indicated mice trea-ted with compound 9# have fewer blood vessels (Fig. 6D).Together, these observations demonstrate that compound 9#treatment exhibited significant anti-tumor capability. As therewas little cytotoxicity of compound 9# on these tumor cells(Fig. 5F), we attributed the anti-tumor activity of the compoundto its capacity to direct and promote the immune system to erad-icate the tumor cells.

3.5. Effects of compound 9# in vivo on TAMs and Treg

According to the in vitro effect of compound 9# on TAMs andTreg, we first analyzed the TAMs’ phenotype separated from com-pound 9#-treated tumors. Compound 9# treatment could increase



Fig. 2. Effect of Compound 9# on the expression of macrophage markers. A–D: Macrophages were pre-incubated with different concentrations of compound 9# for 1 h andthen cultured with LPS (200 ng/ml) or PMA (50 ng/ml) for 24 h. The protein level of IL-10, IL-12/IL-12p40 and TNF-a in the supernatants from peritoneal macrophages (A andB) and THP-1 cells (C and D) were examined by ELISA and the mRNA levels of M1-marker gene NOS2 (A and C) and M2-marker gene Arg1/CCL18 (B and D) in peritonealmacrophages and THP-1 cells were measured by qRT-PCR. E–H: Macrophages were transfected with Jak2-specific siRNA or control siRNA, followed by pre-incubating 10 lMcompound 9# for 1 h and then cultured with LPS (200 ng/ml) or PMA (50 ng/ml) for 24 h. The supernatants from peritoneal macrophages (E and F) and THP-1 cells (G and H)were collected for determination of IL-10, IL-12/IL-12p40 and TNF-a, and gene NOS2 and Arg1/CCL18 expression in peritoneal macrophages (E and F) and THP-1 cells (G andH) were measured by qRT-PCR. I(1, 2) (a, b, c): Macrophages were transfected with Jak2-specific siRNA or control siRNA, followed by pre-incubating 10 lM compound 9# for1 h and then cultured with LPS (200 ng/ml) or PMA (50 ng/ml) for 30 min. The expressions of P-Jak2, T-Jak2, P-STAT3 and T-STAT3 in peritoneal macrophages (I1) and THP-1cells (I2) were evaluated by a Western blot analysis. a, image of western blot; (b, c), grey level of western blot. The results are representative of three independentexperiments and expressed as the means ± SEM. *P < 0.05 compared with the LPS or PMA + 9# (without) group in A–D. *P < 0.05 compared with the LPS or PMA + ControlsiRNA + 9# (without) group in E–H. *P < 0.05 compared with the Control siRNA group in I(1, 2) b. *P < 0.05 compared with the LPS or PMA + Control siRNA + 9# (without)group in I(1, 2) c.


Please cite this article in press as: W. He et al., A Jak2-selective inhibitor potently reverses the immune suppression by modulating the tumor microen-vironment for cancer immunotherapy, Biochem. Pharmacol. (2017), http://dx.doi.org/10.1016/j.bcp.2017.08.019


Fig. 3. Compound 9# induced repolarization of TAMs in vitro via inhibiting Jak2-STAT3 signaling. A-C(1, 2, 3): TAMs were treated with 10 lM compound 9# for 24 h, thesupernatants were harvested for detection of IL-12 and IL-10 (A), gene expression of a subset of M1 and M2markers (NOS2, MHCII, Arg1 and Ym1) in TAMs were measured byqRT-PCR (B) and the expressions of P-Jak2, T-Jak2, P-STAT3 and T-STAT3 were evaluated by aWestern blot analysis (C). D-F (1, 2, 3): TAMs were transfected with Jak2-specificsiRNA or control siRNA, the concentration of IL-12 and IL-10 in the supernatant (D), gene expression of NOS2, MHCII, Arg1 and Ym1 (E) and the P-Jak2, T-Jak2, P-STAT3 and T-STAT3 expression (F) after 10 lM compound 9# treatment. 1, image of western blot; (2, 3), grey level of western blot. The results are representative of three independentexperiments and expressed as the means ± SEM. *P < 0.05 compared with the 9# (without) group in A–C (1, 2, 3). *P < 0.05 compared with the Control siRNA + 9# (without)group in D, E and F3. *P < 0.05 compared with the control siRNA group in F2.


the secretion of the IL-12 and reduce the production of the IL-10(Fig. 7A) in these tumor models. Meanwhile, administration ofcompound 9# also increased M1-specific gene (NOS2 and MHCII)and decreased the expression of M2 marker (Arg1 and Ym1)(Fig. 7B and C). Consistently, in situ immunofluorescence stainingalso demonstrated the same results about the change in theexpression of IL-12 and of IL-10 in the tumor tissues upon com-pound 9# treatment (Fig. 7D and E). We further evaluated theeffect of compound 9# to Treg in vivo and found that compound9# could significantly reduce the proportion of tumor-inducedTreg in the spleen and blood of treated animals (Fig. 7F). These datasuggested a dramatic alteration in the phenotype, number andfunction of TAMs as well as the Treg in compound 9#-treatedtumors, which meant a marked relief to the immunosuppressionfrom these cells.

3.6. Compound 9# increased the proliferation and activity of CD4+ Tcells and CD8+ T cells in vivo

T cells, especially CD4+ and CD8+ T cells, mediated the mostimportant anti-tumor immune reactions in vivo. Most cancerimmunotherapy methods are also aiming to evoke, promote andenhance the specific antitumor activities from CD4+ and CD8+ Tcells. On the other hand, TAMs and Treg also take CD4+ and CD8+

T cells as their targets to suppress the immune rejection to tumorcells through various direct and indirect mechanisms. As com-pound 9# changed the TAMs’ phenotype and function from M2-like to a pro-inflammatory M1-like one and suppressed the gener-ation of Treg in cancer mice, the T cells-mediated anti-tumor


response should be recovered in those compound 9#-treated mice.To address this, we first analyzed CD4+ T cells and CD8+ T cells’populations in each treatment cohort by flow cytometry. In vivoadministration of compound 9# significantly increased the propor-tion of CD4+ and CD8+ T lymphocytes in the blood, spleen andtumor tissues (Fig. 8A–D). In an effort to determine if compound9# treatment re-boosted T cells’ activity against tumor cells, weanalyzed the level of IFN-c in tumor tissues as a measurement ofT-cell function. As shown in Fig. 8E, the expression of IFN-c wasstrongly augmented by compound 9# treatment. Additionally,the outcomes of the immunofluorescence stain for IFN-c correlatedwell with the ELISA results (Fig. 8F). These results indicated thatcompound 9# treated successfully the tumor implanted animalbody to recover the immune system from the immunosuppressivestate to a highly activated anti-tumor state by the enhancement inthe number of functional CD4+ and CD8+ T cells, resulting in theanti-tumor activity of the compound.

4. Discussion

Persistent STAT3 activation is prevalent in immune cells in thetumor environment and contributes to tumor growth [28,29].While direct inhibition of transcription factors such as STAT3 withsmall-molecule inhibitors is still challenging, targeting of upstreamactivating kinases has the potential to become a new approach. Inthis study, we demonstrated that compound 9# is a novel smallmolecule inhibitor of Jak2. Compound 9# belongs to the type ofa, a-difluoro-b-amino-ketones, which contains a trifluoromethylgroup at b-position. It is a white solid and has been demonstrated



Fig. 4. Compound 9# suppressed Treg induction and function in vitro via inhibiting Jak2 activation. A and B: Naive CD4+ T cells were stimulated with anti-CD3, anti-CD28monoclonal antibodies and TGF-b (2 lg/ml) under the indicated doses of compound 9# and analyzed by FACS. C: CD4+ CD25+ Treg were treated with the indicatedconcentrations of compound 9# for 24 h, and supernatants were collected for determination of IL-10 and TGF-b. D (1, 2, 3): Immunoblotting of P-Jak2, T-Jak2, P-STAT3 and T-STAT3 in CD4+ T cells under Treg-inducing conditions and treated with the indicated concentrations of compound 9#. E and F: CD4+ T cells were transfected with Jak2-specificsiRNA or control siRNA, followed by were stimulated with anti-CD3, anti-CD28 monoclonal antibodies and TGF-b (2 lg/ml) under 10 lM compound 9# and analyzed by FACS.G: CD4+ CD25+ Treg were transfected with Jak2-specific siRNA or control siRNA, treated with 10 lM compound 9# for 24 h, and supernatants were collected fordetermination of IL-10 and TGF-b. H (1, 2, 3): Immunoblotting of P-STAT3, T-STAT3 and T-Jak2 in CD4+ T cells under Treg-inducing conditions and treated with 10 lMcompound 9# after silencing of Jak2 by Jak2-specific siRNA. 1, image of western blot; (2, 3), grey level of western blot. The results are representative of three independentexperiments and expressed as the means ± SEM. *P < 0.05, and **P < 0.01 compared with the 9# (0 lM) group in B, C and D (2, 3). *P < 0.05 compared with the 9# (0 lM)+ control siRNA group in F, G and H3. *P < 0.05 compared with the control siRNA group in H2.




Fig. 5. Compound 9# treatment significantly retarded tumor growth. A and B: (A) Mean tumor weights on two weeks post-first time at different doses of compound 9#treatments and (B) tumor growth curves from mice receiving intratumoral injections of compound 9#. C: Survival rate of tumor-bearing mice treated with differentconcentrations of compound 9#. D: Representative images of tumor harvested frommice in each treatment cohort. Scale bar, 1 cm. E: Tumor sections frommice that receiveddifferent doses of compound 9# treatments were examined by H&E staining. Scale bar, 100 lM. F: The viability of B16F10 and 4T1 cells with different concentrations ofcompound 9# for 72 h, the data were normalized to DMSO treated cells. Tumor cells were mixed TAM treated with or without compound 9# and then injected into mice. G:Mean tumor weights after mixing tumor cells with TAM treated with or without compound 9# injected into mice for 20 days. H: tumor growth curves from mice injectedtumor cells with TAM treated with or without compound 9#. I: Representative images of tumor harvested from mice in each treatment cohort. Scale bar, 1 cm. The values areexpressed as the means ± SEM (N = 8 mice/group). *P < 0.05 compared with the PBS group in A and B. *P < 0.05 compared with the tumor plus TAM group in G and H.


to be configurational stable. It can be easily deprotected and con-verted to more functionally complex derivatives such as b-aminoacids [20]. Meanwhile, a, a-difluoro carbonyl compounds are


known to form relatively stable hydrates, a feature that makesthem become the inhibitors of a wide range of enzymes suchas a-chymotrypsin and HIV-1 protease [30,31]. Therefore,



Fig. 6. Reprogramming of TAMs and inhibiting Treg generation by compound 9# caused a reduction in tumor angiogenesis. A: The protein level of MMP-9 and VEGF intumors harvested from mice with different doses of compound 9# treatments was tested by ELISA. B and C: Immunofluorescent staining for MMP-9 and VEGF in B16F10 and4T1 tumor tissues from mice treated with different concentrations of compound 9#. D: Immunofluorescent staining of CD31 was used to determine the amount ofangiogenesis in mice treated with different concentrations of compound 9#. Red, MMP-9/VEGF/CD31; blue, DAPI nuclear staining. Scale bar, 100 lm. N = 8 mice per group.The values are expressed as the means ± SEM (N = 8 mice/group). *P < 0.05 compared with the PBS group in A. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)


compound 9# may have a greater potential to be developed as apharmaceutical for cancer immunotherapy.

STAT3 signaling is also implicated in the differentiation ofdefective dendritic cells (DC) as well as the activation and expan-sion of MDSCs, in addition to its role in promoting the M2 polariza-tion of macrophages and the proliferation and immunosuppressivefunction of Treg. Studies have demonstrated that hyper-activationof Jak2-STAT3 pathway directly mediates the abnormal differenti-ation of DC and causes accumulation of immature myeloid cells incancer [32]. Inhibition of Jak2-STAT3 pathway could cause dra-matic activation of DCs and enhance their ability to stimulateantigen-specific T-cell response such as JSI-124 and cucurbitacin[33–35]. MDSCs from tumor-bearing mice showed higher levelsof activated STAT3 than the immature myeloid cells from naivemice did [36]. Inhibition of STAT3 by selective STAT3 inhibitor orthe use of conditional knockout mice could obviously reduce theexpansion of MDSCs, and restore T-cell responses in tumor-bearing mice [37]. Moreover, engineering STAT3-silenced CD8+ Tcells or targeting STAT3 systemically with small-molecule inhibi-tors could increase transferred CD8+ T cell infiltration and activa-tion at the tumor site [38]. Therefore, the inhibition of STAT3signaling to modulate the tumor environment is regarded as oneof the promising strategies for the development of new anti-tumor therapeutics.

Previous studies mainly focused on impairing the Jak-STAT3signaling by small molecules within tumor cells using in vitroand immunocompromised xenograft models [39,40]. AZD1480, a


potent, competitive small molecule inhibitor of Jak1/2 kinase,could suppress the growth of human solid tumor xenografts in aSTAT3-dependent manner [14]. 6-Bromoindirubin-30-oxine, apan-Jak inhibitor, could induce apoptosis of human melanomacells in vitro and inhibit tumor growth in vivo with low toxicityin a mouse xenograft model of melanoma [41]. Interestingly, insome tumor cells such as glioma tumor cells, ovarian carcinoma(OC) cells and melanoma cells, AZD1480 did not inhibit tumor cellgrowth in vitro, but the tumor growth was suppressed in vivo ineach tumor model with AZD1480 treatment [42–44]. In the trans-genic mouse model of OC, AZD1480 could inhibit tumor-associatedMMP activity, and decrease suppressor T cells (Treg) generation inthe peritoneal tumor microenvironment. In addition, AZD1480could also inhibit angiogenesis and metastasis in a human xeno-graft tumor model [45]. In agreement with these findings, our datashowed that compound 9# had little effect on cell viability ofcultured B16F10 and 4T1 cells, but could retard tumor growthin vivo. Our results also showed that compound 9# successfullyreactivated tumor-associated macrophages and inhibited Treggeneration in vitro and in vivo, and inhibited tumor-associated pro-tease activity and angiogenesis, leading to the reversion of tumormicroenvironment and suppression of tumor growth.

In conclusion, compound 9# is a novel small molecule inhibitorof the Jak2 kinase. Through the inhibition of Jak2-STAT3 signaling,compound 9# acted to re-direct TAMs’ polarization and inhibitTreg induction both in vitro and in vivo, thereby restoring immunesurveillance against tumor. These promising findings warrant



Fig. 7. Compound 9# altered TAMs phenotype and suppressed Treg in vivo. A–C: TAMs purified from tumor-bearing mice treated with different concentrations of compound9#, and cultured in RPMI-1640 medium containing 10% FBS for 24 h. The concentrations of IL-12 and IL-10 in the supernatant were examined by ELISA (A) and NOS2, MHCII,Arg1 and Ym1 mRNA expression levels were determined by qRT-PCR (B and C). D and E: Immunofluorescence of tumor sections was also performed to examine theexpression of IL-12 and IL-10 in tumor tissues. Red, IL-12/IL-10; blue, DAPI nuclear staining. Scale bar, 100 lm. F (1, 2) (a, b, c): The frequency of Treg (F1a and F2a) andrepresentative flow cytometry analysis (F (1, 2) (b, c)) in spleen and tumor tissues after compound 9# treatment. The results are expressed as the means ± SEM (N = 8mice/group). *P < 0.05 compared with the PBS group in A–C and F (1, 2) (b, c). (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)




Fig. 8. Compound 9# restored immunosurveillance against tumor in vivo. A, B (1, 2, 3), C and D (1, 2, 3): The frequency of CD4+ T cells and CD8+ T cells (right) andrepresentative flow cytometry analysis (left) in blood, spleen and tumor tissues after compound 9# treatment. E: Quantification of the IFN-c level in the tumor tissues byELISA. F: Immunofluorescent staining for IFN-c in the tumor tissues from mice treated with PBS, DMSO or different doses of compound 9#. Green, IFN-c; blue, DAPI nuclearstaining. Scale bar, 100 lm. The results are expressed as the means ± SEM (N = 8 mice/group). *P < 0.05 compared with the PBS group in B (1, 2, 3), D (1, 2, 3) and E. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)





further investigations for the development of this compound into anew, potent, small molecule agent for cancer immunotherapy.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgment

This project was funded by the National Basic Research Programof China (2012CB517603), the National High Technology Researchand Development Program of China (2014AA020707), the NationalNatural Science Foundation of China (31271013, 31170751,31200695, 31400671, 51173076, 51503232, 91129712 and81102489), the Program for New Century Excellent Talents inUniversity (NCET-13-0272), Nanjing University State Key Labora-tory of Pharmaceutical Biotechnology Open Grant (02ZZYJ-201307). C.W. acknowledges the funding supports from Universityof Macau (MYRG2015-00160-ICMS-QRCM) and the opening fundof the State Key Laboratory of Quality Research in Chinese Medi-cine, University of Macau (No. 005).

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