Polarization of tumor-associated macrophage is associated with tumor vascular normalization by endostatin

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1 Thorcic Cncer ISSN ORIGINAL ARTICLE Polriztion of tumor-ssocited mcrophge is ssocited with tumor vsculr normliztion y endosttin Qin Peng 1 *, Mei Li 1 *, Zi Wng 1, Ming Jing 1,XiYn 1, Song Lei 2, Hui Zhng 1, Wei Zhng 1, Yn-Yng Liu 1 * & Feng Luo 1 1 Deprtment of Medicl Oncology, Cncer Center nd Stte Key Lortory of Biotherpy, West Chin Hospitl of Sichun University, Chengdu, Sichun Province, Chin 2 Deprtment of Pthology, West Chin Hospitl of Sichun University, Chengdu, Sichun Province, Chin Keywords Endosttin; normliztion; tumor vsculture; tumor-ssocited mcrophges. Correspondence Feng Luo, Deprtment of Medicl Oncology, Cncer Center nd Stte Key Lortory of Biotherpy, West Chin Hospitl of Sichun University, 37 Guoxue Xing Street, Chengdu , Sichun Province, Chin. Tel: Fx: Emil: luofeng@medmil.com.cn Received: 25 Octoer 2012; ccepted 30 Novemer doi: / *These uthors contriuted eqully to this work. Astrct Bckground: Vsculr normliztion is n emerging concept in cncer tretment, ut its precise mechnisms re not completely understood. The polriztion of tumor-ssocited mcrophges (TAMs) is importnt in tumor ngiogenesis nd metstsis. However, little is known out the effect of nti-ngiogenic gents on the polriztion of tumor-ssocited mcrophges. Therefore, we explore the chnges of TAMs polriztion in the development of tumor vsculr normliztion induced y endosttin. Methods: A murine xenogrft model of lung cncer ws treted with endosttin for 10 dys. The morphology nd function of tumor vsculture ws exmined using vrious techniques. Flow cytometry ws crried out to ssess the TAMs, nd immunofluorescence ws used to exmine Tie-2-expressing monocytes (TEMs) in tumors. Levels of the histidine-rich glycoprotein (HRG) in tumors were mesured y immunohistochemistry nd Western lot. Results: Tumor vessels ecme more norml nd mture on dy six in the endosttin-treted mice. During vsculr normliztion, the numer of M2-like TAMs nd TEMs in the tumors ws significntly reduced, wheres the numer of M1-like TAMs showed n increse on dy six fter endosttin tretment, lthough the ltter ws not sttisticlly significnt. The HRG in the tumors ccumulted t n erly stge fter endosttin dministrtion. Conclusions: The polriztion of TAMs is ssocited with tumor vsculr normliztion induced y endosttin. These oservtions my e useful in the explortion of new strtegies for nti-ngiogenic tretment. Introduction The growth nd metstsis of solid tumors is highly dependent on their ngiogenesis. However, tumor lood vessels re structurlly nd functionlly norml; they re disorgnized, dilted, nd tortuous, with excessive rnching nd shunts, uneven dimeters, nd high permeility. 1 The resulting chotic nd vrile lood flow impedes the delivery of ntitumor gents into the tumors. Moreover, these normlities crete hypoxic nd cidic tumor microenvironment, which ttenutes the effects of mny nticncer therpies, such s chemotherpy,rdiotherpy,nd immunotherpy. 2 Jin hypothesized tht certin nti-ngiogenic gents could trnsiently normlize the norml structure nd function of tumor vsculture to mke it more efficient for oxygen nd drug delivery. 3 In recent yers, ccumulting dt hs confirmed tht mny nti-ngiogenic gents cn led to tumor vsculr normliztion, 4 7 which hs lid theoreticl foundtion for optiml scheduling of ntitumor gents, when comined with other nti-tumor strtegies. The vessel normliztion induced y nti-ngiogenic gents is gining more nd more ttention in the field of cncer reserch. However, the detiled mechnisms of this normliztion remin elusive. Tumor-ssocited mcrophges (TAMs) re heterogeneous cell popultion originting from the mononucler phgocytic linege. 8,9 In vriety of solid tumors, the infiltrted TAMs re the mjor components of inflmmtory cells Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd 295

2 The mechnism of tumor vsculr normliztion Q. Peng et l. in the tumor microenvironment, nd their ccumultion is considered to e relted to the tumor growth nd metstsis Diversity nd plsticity re hllmrks of TAMs, which cn e polrized to different phenotypic sugroups in different microenvironmentl conditions. 13 The clssiclly ctivted (M1-like) TAMs reduce ngiogenesis nd increse inflmmtion, ut the lterntively ctivted (M2- like) TAMs usully promote tumor growth nd stimulte ngiogenesis. 14,15 Recently, Rolny 16 showed tht deposited histidine-rich glycoprotein (HRG) in tumor strom cn normlize tumor vessels y skewing TAM polriztion from n M2-like phenotype to n M1-like phenotype. This study not only provides unprecedented insight into the centrl role of TAM polriztion in tumor vsculr normlities nd tumor metstsis, it lso offers etter understnding of the mechnisms of tumor vsculr normliztion. 17 Severl lines of direct nd indirect evidence indicte tht some nti-ngiogenic gents medite TAMs infiltrtion in vriety of solid tumors. 18,19 However, little is known out the effect of nti-ngiogenic gents on the polriztion of TAMs. Endosttin, rod-spectrum endogenous ngiogenesis inhiitor, cn induce tumor vsculr normliztion to some extent, s confirmed y our, nd other, studies. 7,20 22 Until now, unfortuntely, no informtion hs een cquired concerning the influence of endosttin on TAMs polriztion during tumor vsculr normliztion. In the present study, we extend our reserch to explore the role of TAMs polriztion in the development of tumor vsculr normliztion induced y endosttin. We estlished mouse model of lung cncer nd treted the mice with endosttin, s previously reported. We found tht endosttin normlized tumor vessels clerly, nd promoted mcrophge infiltrtion. The polrity of TAMs chnged to n M1-like phenotype during the tumor vsculr normliztion. At sme time, more HRG ws found to e expressed in the tumor t the erly stge fter endosttin dministrtion. Methods Cell culture nd nimls Humn lung denocrcinom cell line SPC-A1 cells (Chinese Acdemy of Sciences, Shnghi, Chin) were mintined in RPMI-1640 medium supplemented with 10% fetl ovine serum nd kept in humidified 5% CO 2 tmosphere incutor t 37 C. Femle thymic nude mice (6 to 8 weeks old) were purchsed from HFK Bioscience Compny (Beijing, Chin). The mice were red in the Sichun University Lortory Animl Reserch Center in pthogen-free conditions nd kept on 12-h light/drk cycle with food nd wter d liitum. The temperture in the rooms ws mintined t 25 1 C nd the reltive humidity mintined t 40 60%. All studies involving mice were crried out in ccordnce with guidelines of the Lortory Animl Cre Committee of Sichun Province (licence no. SYXK ). Tretment protocol Mice were sucutneously implnted with 100 ml solution contined SPC-A1 cells in the right mid-dorsl flnk. Tumor volume ws evluted directly with clipers nd estimted using the formul: tumor volume (mm 3 ) = 0.52 length width. 2 When tumor volume reched out 200 mm 3, the mice were rndomly ssigned to the endosttin group (ES group) or sline control group (NS group). ES group mice were given 3 mg/kg endosttin y dily cudl vein injection (recominnt humn endosttin; Simcere Medgenn Bio-Phrmceuticl Compny, Chin) for 10 dys, nd the NS group received 0.9% sline vi vein s control. Six mice from ech group were scrificed t three time points (t dys three, six, nd 10 fter the initition of tretment) to hrvest lood smples nd tumor tissues for reserch. Histology, immunohistochemistry, nd immunofluorescence Tumor tissues were rpidly frozen t 80 C or fixed with formlin nd emedded in prffin. Prffin sections were routinely stined with hemtoxylin nd eosin (H&E) for pthologic histology exmintion. Hlf of the prffin sections (thickness 3 5 mm) were used to detect HRG, nd the djcent frozen sections (thickness 6 8 mm) were used to detect expression of CD31, lph smooth muscle ctin (-SMA), Tie-2, nd F4/80. Sections were incuted t 4 C overnight with one of the following primry ntiodies: rit nti-mouse Tie-2 (1:200, Snt Cruz, CA, USA); rt nti-mouse F4/80 (1:400, Snt Cruz, CA, USA); rit ntimouse HRG (1:400, Acm, Cmridge, MA, USA); rit nti-mouse -SMA (1:200, Acm, Cmridge, MA, USA); or rt nti-mouse CD31/PECAM (1:400, BD Phrmingen, Sn Jose, CA). The sections were then incuted with pproprite secondry ntiodies (Snt Cruz, CA, USA) for immunofluorescence or immunohistochemistry. Nuclei were highlighted using 4,6-dimidino-2-phenylindole (DAPI; 1:1000, Invitrogen) for five minutes. Microvessel density (MVD) ws evluted. 23 The pericyte coverge ws estimted y quntitting the percentge of microvessels tht coloclized endothelil cell stining (CD31) nd pericyte stining (-SMA). It ws ssessed in nine rndom fields (three mice per group) t mgnifiction ( 200) for sttistics. Sections were viewed nd digitlly photogrphed using fluorescence microscope (Olympus UIS2). Trnsmission electron microscopy Specimens were prepred for trnsmission electron microscopy. 24 SPC-A1tumorsweretrimmedtomximldimension 296 Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd

3 Q. Peng et l. The mechnism of tumor vsculr normliztion of 1 mm. The excised tumors were fixed with 1% glutrldehyde nd 4% formldehyde in 0.1 M phosphte uffer (ph 7.4) for minimum of two hours, post-fixed in 1% osmium tetroxide for 30 minutes, dehydrted in cetone, nd emedded in epoxy resin. For histologicl nlysis, sections of 1.0 mm were stined with toluidine lue. Seventy nm sections were cut with microtome (Leic Microsystems, Vienn, Austri) nd stined with led citrte nd urnyl cette. The intrtumorl vsculture ws visulized using n H7650 trnsmission electron microscope (Hitchi,Tokyo,Jpn). Detection of tumor hypoxi An immunofluorescence detection kit ws used. 25 In rief, 60 mg/kg of pimonidzole ws injected vi the cudl vein 20 minutes efore the mice were scrificed. The Hypoxyproe-1 kit (Hypoxyproe, Inc. HPI Burlington, MA, USA), consisting of 100 mg solid pimonidzole HCl nd 1.0 ml fluorescein isothiocynte (FITC)-leled mouse IgG1 monoclonl ntiody ginst protein dducts of pimonidzole, ws used to detect pimonidzole protein dducts in tumor sections. Tumor tissue ws hrvested nd rpidly frozen t 80 C nd the frozen sections (thickness 6 8 mm) were used for oservtion of hypoxic regions. Flow cytometry Flow cytometry ws used to distinguish the different sugroups of TAMs. Collgense IV (Gico, USA) ws dissolved in serum-free medium nd the finl concentrtion ws 1 mg/m. Single-cell suspensions from the tumors of scrificed nimls were prepred. 26 The following fluorochrome conjugted ntiodies were used: FITC-leled rt ntimouse CD206, Alex Fluor488-leled rt nti-mouse CD197, phycoerythrin (PE)-leled rt nti-mouse CD68 (Biolegend, Sn Diego, USA), nd the pproprite isotype control ntiodies (Biolegend, Sn Diego, USA). Cells were incuted with ntiodies t 4 C for 30 minutes. Flow cytometry ws performed with flow cytometer (BD FACSCliur; BD Biosciences), with the nlysis gtes designed to delinete the group of mcrophges. Western lot nlysis Fresh tumor tissue ws put into tues nd immersed in liquid nitrogen s soon s possile. Tissue ws crushed while frozen in grinding mortr y shking with sterile grinding rods. The tissue powder ws crcked in lysis uffer (Beyotime, Chin) nd then homogenized with n ultrsonic homogenizer. After centrifugtion t rpm for 15 minutes t 4 C, equl mounts of protein from ech group were seprted y SDS-PAGE. The seprted proteins were trnsferred to PVDF memrnes (Millipore) y electrolotting. The memrnes were locked in 5% non-ft dried milk in TBST (20 mm Tris, 500 mm NCl, 0.1% Tween-20, ph 7.6), nd incuted overnight t 4 C with primry rit nti-mouse HRG ntiody (1:1000, Acm, Cmridge, MA, USA). The memrne ws then incuted with n pproprite secondry ntiody conjugted with horserdish peroxidse. Rective nds were detected y the enhnced chemiluminescence (Amershm Biosciences Corp, Pisctwy, NJ, USA). Sttisticl nlysis Dt is presented s mens stndrd devition (SD). Sttisticl comprison ws performed using the Sttisticl Pckge for the Socil Science (SPSS) version 13.0 (Chicgo, Illinois, USA). We used the unpired Student s t-test for microvsculr density nd the numer of F4/80 + cells, nd the rnk sum test for percentge comprisons. P-vlues <0.05 were considered to e significnt. Results Endosttin promotes vsculr normliztion nd improves tumor perfusion Becuse endosttin is recominnt humn protein nd the SPC-A1 is humn lung denocrcinom cell line, we chose thymic nude mice s n experimentl niml to void immunologicl rection to the extrinsic protein ntigen nd the tumor xenogrft To exmine the morphology nd function of tumor vsculture, we used vriety of methods to detect vsculr chnges. In our recent study, we found tht the time window of vsculr normliztion is pproximtely three to six dys fter endosttin dministrtion. 7 Therefore, in this study, six mice from ech group were scrificed t three time points (t dys three, six, nd 10 fter initition of the tretment) for exmintion. We found tht compred with the NS group, on dys three, six, nd 10, fter the dministrtion of tretment, the MVD in the ES group reduced to 72.7%, 36.9%, nd 44.8% of tht efore tretment, respectively (P < 0.01; Fig. 1A,B). Pericyte coverge ws quntified y doule immunofluorescent stining of frozen sections with CD31 nd -SMA. On dy six, there ws significnt difference in pericyte coverge etween the ES group nd the NS group ( % vs %; P < 0.01; Fig. 1C nd 1D). The rchitecture of intrtumorl vessels ws exmined y trnsmission electron microscopy. We lso found tht the pericytes showed swollen, irregulr shpe with utophgic odies in the cytoplsm, nd the tumor vessel lumen tended to close up in the NS group (Fig. 2A,B). The vsculr sement memrne ws closely ssocited with endothelil cells during endosttin dministrtion. Tumor vsculr wlls nd pericytes were more regulr, nd red lood cells ppered in the lumen fter endosttin tretment (Fig. 2C,D). Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd 297

4 The mechnism of tumor vsculr normliztion Q. Peng et l. Figure 1 Endosttin reduces microvessel density (MVD) nd increses pericyte coverge in SPC-A1 xerogrphs. When the implnted tumor reched volume of out 200 mm 3, the mice in the endosttin group were treted y cudl vein injection with endosttin 3 mg/kg dily for 10 dys, nd the mice in the control group were treted with n equl volume of 0.9% sline. On dys three, six, nd 10, fter the initition of tretment, mice (n = 6) were scrificed. () Photogrphs represented the CD31 in SPC-A1 crcinom on dys three, six, nd 10, respectively. Originl mgnifiction: 200. () Quntittive comprison of MVD. (c) The pericyte coverge of intrtumorl vsculr wlls on dy six ws represented y immunofluorescence. Originl mgnifiction: 200. (d) The percentge of endothelil cells covered y pericytes. Dt is exhiited s men stndrd devition (SD), columns, mens; rs, SD;*P < 0.05; **P < 0.01; ***P < 0.005, compred with control group., NS;, ES. The res of hypoxi nd necrosis were ssessed to determine tumor perfusion.nine fields(three mice per group) were rndomly chosen for evluting hypoxic nd necrotic res. The frctions of hypoxic nd necrotic res on the tumor sections were digitlly nlyzed y ImgeJ, s in previous studies. 21 FITC fluorescence-positive res suggest hypoxic regions,which re distriuted s irregulr regions in the tumor prenchym.at dy six fter endosttin tretment,the hypoxic res were mrkedly reduced ( % in ES group vs % in the NS group; P < 0.01; Fig. 3A,B), nd the necrotic res were similrly reduced ( % in ES group vs % in the NS group; P < 0.005; Fig. 3C,D). Overll, these results show tht endosttin improved tumor vessel normliztion nd mturtion. Endosttin promotes mcrophge infiltrtion in SPC-A1 xenogrfts Previous studies suggest tht nti-ngiogenic gents ffect mcrophge infiltrtion in solid tumors. 18,19 However, there hs een no evidence confirming tht endosttin could impct on mcrophge infiltrtion. Therefore, we investigted the infiltrtion of TAMs in the tumor microenvironment. Immunofluorescence ws performed using mcrophge mrkers F4/80 to exmine the numer of TAMs on three, six, nd 10 dys. Our results showed tht F4/80 + mcrophges were significntly incresed on dy 10 fter endosttin tretment ( in ES group vs in NS group; P < 0.05; Fig. 4A,B). Endosttin ffects the polriztion of tumor-ssocited mcrophges Evidence suggests tht TAMs frequently show n M2-like phenotype, which cn promote ngiogenesis. 13 Moreover, reducing the M2-like polriztion of TAMs cn lso induce normliztion of tumor vsculture. 17 We,therefore,explored the effects of endosttin on the polriztion of TAMs. In this experiment,weusednnimlmodelof thymicnudemiceto oserve the chnges of mcrophges, s other reserchers hve 298 Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd

5 Q. Peng et l. The mechnism of tumor vsculr normliztion c d Figure 2 Endosttin normlizes the rchitecture of tumor vsculr wlls. Trnsmission electron microscopy imges represent tumor lood vessels on dy six fter the initil tretment. () nd () show the vsculr rchitecture in the tumor of the control group. (c) nd (d) represent the tumor vsculr wlls from the endosttin group. Originl mgnfiction for () nd (c), 0.7k; originl mgnifiction for () nd (d), 1.5k; RBC, red lood cells. studied The polrity of TAMs ws ssessed y the expression of mrker of the mcrophge linege (CD68), nd prticulr mrkers of the sugroups of this linege(cd197 for the M1-likesugroupndCD206fortheM2-likesugroup).Representtive quntifictions of the proportions of M2-like nd M1-like mcrophges otined y flow cytometry re shown in Figure 5A nd B. We found tht the verge percentge of M2-like TAMs in the ES group ws significntly lower on dy six ( % in the ES group vs % in the NS group;p<0.05;fig. 5C),ut then reounded on dy 10 ( % in ES group vs % in NS group; P < 0.05; Fig. 5C).The percentge of M1-like TAMs incresed on dy six fter endosttin tretment (Fig. 5D), ut this ws not sttisticlly significnt. These findings show tht endosttin skewed Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd 299

6 The mechnism of tumor vsculr normliztion Q. Peng et l. c d Figure 3 Endosttin improves oxygention nd perfusion of SPC-A1 crcinom. () Representtive photogrphs of tumor tissue hypoxi t different time points. Originl mgnifiction: 40. () Histogrm of men hypoxic frction on three, six, nd 10 dys. (c) Imges of necrosis in SPC-A1 crcinom fter tretment. Originl mgnifiction: 100. The lck rrows indicte the necrotic res in the tumor. (d) Histogrm of men necrosis frction t the three time points. The tumor hypoxi nd necrosis ws significntly relieved on dy six fter endosttin tretment. Dt is expressed s men stndrd devition (SD), columns, mens; rs, *P < 0.05; **P < 0.01; ***P < 0.005, compred with the control group. TAM polriztion wy from the M2-like phenotype during tumor vsculr normliztion. Endosttin reduces numers of Tie-2-expressing monocytes Tie-2-expressing monocytes (TEMs) re suset of tumorinfiltrting myeloid cells with pro-ngiogenic function. 33 Some studies suggest tht TEMs hve chrcteristics of M2-like TAMs Therefore, we exmined the numer of TEMs in the tumors. We distinguished Tie-2-positive cells from the tumor-infiltrting F4/80 + mcrophges y doulestining immunofluorescence. We found tht Tie-2- expressing mcrophges were clerly reduced on dy six fter endosttin tretment ( % in ES group vs % in NS group; P < 0.05; Fig. 6A,B). HRG ccumulted following dministrtion of endosttin HRG, host-produced multi-domin plsm protein, hs n importnt role in the regultion of tumor ngiogenesis nd cn normlize tumor vessels y polrizing TAMs. 16 Therefore, we exmined the expression of HRG protein deposited in the tumors. An immunohistochemistry ssy demonstrted tht HRG protein expression in the tumor in the ES group ws higher thn tht in the NS group (Fig. 7A). In ddition, Western lot nlysis lso showed higher expression of HRG in the tumor on three nd six dys fter endosttin tretment (Fig. 7B). These results indicte tht more HRG is deposited in the tumor in the erly stges fter endosttin dministrtion. 300 Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd

7 Q. Peng et l. The mechnism of tumor vsculr normliztion Figure 4 Endosttin promotes mcrophge infiltrtion. Sections of tumors from the control group nd endosttin group mice were evluted y immunofluorescence for mcrophge mrkers. () Typicl photogrphs represent the F4/80 + cells (red) t the three time points. Blue, DAPI-stined nuclei. Originl mgnifiction: 200. The white rrows point to the representtive F4/80 + cells. () F4/80 + cells in tumors from endosttin-treted mice showed significnt increse (*P < 0.05), compred with 0.9% sline-treted mice on dy 10 fter initition of tretment. The numer of the F4/80 + cells ws determined y summing the positive stining res in nine rndom fields t 200. c d Figure 5 Flow cytometric nlysis for the polriztion of tumor-ssocited mcrophges (TAMs). Single cell suspensions were generted from tumor tissues on three, six nd 10 dys fter initil tretment. Then the M2 nd M1-like TAMs were enumerted y two-color flow cytometry, using pnel of monoclonl ntiodies recting with murine CD68 (pn-mrker of tissue mcrophges) nd murine CD206/murine CD197. () Flow cytometry for M2-like TAMs. () Flow cytometry for M1-like TAMs. (c) The verge percentge of M2-like TAMs. (d) The verge percentge of M1-like TAMs. Dt is expressed s men stndrd devition (SD), columns, mens; rs, *P < 0.05;**P < 0.01; ***P < 0.005, compred with the control group. Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd 301

8 The mechnism of tumor vsculr normliztion Q. Peng et l. Figure 6 Effects of endosttin on Tie-2-expressing monocytes (TEMs). Fluorescein isothiocynte (FITC) green fluorescence nd Texs Red fluorescence represent the Tie-2-positive cells nd F4/80-positive cells, respectively. () The merged fluorescence photogrphs represent the TEMs in SPC-A1 xenogrfts. The white rrows refer to the representtive TEMs. Originl mgnifiction: 400. () The percentge of the F4/80 + cells which express Tie-2 ws determined y summing the positive stining res in nine rndom fields t 400. Dt is expressed s men stndrd devition (SD), columns, mens; rs, *P < 0.05; **P < 0.01; ***P < 0.005, compred with the control group. Discussion Vsculr normliztion is n emerging concept in cncer tretment. Mny nti-ngiogenic gents cn led to tumor vsculr normliztion. The trnsient normliztion of Figure 7 Effects of endosttin on histidine-rich glycoprotein (HRG) expression. () Detection of HRG expression in tumors y immunohistochemistry in SPC-A1 xenogrfts on dy six fter initil tretment. Originl mgnifiction: 400. The yellow rrows indicte the positive stining regions of HRG. () Western lot nlysis of the HRG expression in SPC-A1 xenogrfts on dys three, six, nd 10 fter initil tretment. The internl reference ws presented y GAPDH. tumor vsculture increses the rte of delivery of drugs nd oxygen into the tumor nd enhnces the effects of chemotherpy nd rdiotherpy. 4 7,21,22 However, the exct mechnism y which endosttin induces the normliztion of tumor vsculture still needs to e fully elucidted. Here, we exmined the effects of endosttin on the structure nd function of tumor vsculture, the relted cells, nd proteins in lung cncer xenogrft murine model. Endosttin trnsiently normlized the morphology nd function of tumor vsculture. The mechnisms of this vsculr normliztion might involve the polriztion of TAMs, lower numers of TEMs, nd higher levels of expression of HRG in the tumor. These suggestions were supported y our results. There ws significnt reduction of in MVD, nd in the re ffected y hypoxi nd necrosis following endosttin dministrtion. The percentge of pericyte coverge ws incresed y endosttin trnsiently, nd the tumor vsculr wlls nd pericytes ecme more regulr. The polrity of TAMs tended to skew towrds M1-like phenotype during vsculr normliztion. In ddition, more HRG ccumulted in tumors t n erly stge fter endosttin dministrtion. In norml tissues, lnce etween pro-ngiogenic nd nti-ngiogenic fctors mintins the norml structure of the vsculture. In tumor tissues, tumor vessels grow in circumstnces where the lnce is tipped towrds the prongiogenic side. Vsculr normliztion will occur when the new lnce etween pro- nd nti-ngiogenic molecules is estlished gin. 37,38 Accumulting evidence indictes tht the collortive ction of pro-ngiogenic fctors (e.g., VEGF, FGF, nd Ang-2) nd endogenous nti-ngiogenic or vsculr stilizing fctors (e.g., TSP-1 nd Ang-1) contriute 302 Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd

9 Q. Peng et l. The mechnism of tumor vsculr normliztion to the vsculr normliztion induced y nti-ngiogenic therpy. 21,39,40 In recent yers, genetic nlysis hs lso demonstrted tht some genes (e.g., Ang-2, PHD2, nd Rgs5) tht determine the mturtion of tumor vessels re involved the normliztion progress More recently, it is suggested tht polriztion of TAMs is novel strtegy for the normliztion of tumor vsculture. 17 TAMs represent dominnt myeloid popultion in mny solid tumors, nd their ccumultion correltes with poor prognosis. However, it is not only their numers, ut lso their phenotype tht regultes tumorigenesis nd ngiogenesis. 14 Recently, Rolny 16 hs reported tht tumor growth nd metstsis re inhiited y the induction of mcrophge polriztion nd vessel normliztion y down regultion of plcentl growth fctor, which suggested tht the trgeting of TAM polriztion could e novel strtegy for vsculr normliztion. In this study, we found tht endosttin ffects infiltrtion nd the polriztion of TAMs, nd tht numers of TEMs, suset of tumor-infiltrting myeloid cells with chrcteristics of M2-like TAMs, re significntly reduced during tumor vsculr normliztion. Our findings provide further evidence to support the importnce of TAMs polriztion in development of tumor vsculr normliztion. Endosttin, 20-kD C-terminl gloulr domin of collgen XVIII, is rod-spectrum nti-ngiogenic gent. Its precise moleculr mechnisms of nti-ngiogenesis remin incompletely understood, lthough numer of inding proteins, such s integrins, tropomyosin, glypicns, lminin, nucleolin, nd MMP2, hve een reported s endosttin receptors In the present study, we found tht endosttin skewed TAMs polriztion wy from the M2-like phenotype during tumor vsculr normliztion. Do TAMs express ny receptor for endosttin? Wht is the signl pthwy tht is responsile for endosttin-induced polriztion of TAMs? A series of further studies must e explored to nswer these questions. HRG, multi-domin plsm protein deposited in the tumor, is synthesized y heptocytes nd hs n importnt function in the regultion of tumor ngiogenesis. 47,48 Emerging evidence indictes tht HRG cn normlize tumor vessels y polrizing TAMs. 16 In this study, we found tht HRG ccumulted in the tumor t n erly stge fter endosttin dministrtion. It is uncler whether or not the influence of endosttin on TAMs polriztion for the tumor vsculr normliztion is HRG-dependent. Further studies re needed to demonstrte the reltionship etween endosttin nd HRG in the tumor microenvironment. Conclusion In conclusion, our dt in the present study indictes tht there re more M1-like TAMs nd HRG, ut less M2-like TAMs ccumulted in the tumor fter endosttin dministrtion. The polriztion of TAMs is ssocited with tumor vsculr normliztion induced y endosttin. These oservtions provide new wy to understnd the mechnisms of vsculr normliztion, nd my e useful in further explortion of new strtegies for nti-ngiogenic tretment. Acknowledgment This study ws supported y the Ntionl Nturl Science Foundtion of Chin (No , no ). Disclosure sttement No uthors report ny conflict of interest. References 1 Crmeliet P, Jin RK. Angiogenesis in cncer nd other diseses. Nture 2000; 407: Fukumur D, Jin RK. Tumor microenvironment normlities: cuses, consequences, nd strtegies to normlize. J Cell Biochem 2007; 101: Jin RK. Normlizing tumor vsculture with nti-ngiogenic therpy: new prdigm for comintion therpy. Nt Med 2001; 7: Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jin RK. Vsculr normliztion y vsculr endothelil growth fctor receptor 2 lockde induces pressure grdient cross the vsculture nd improves drug penetrtion in tumors. Cncer Res 2004; 64: Btchelor TT, Sorensen AG, di Tomso E et l. AZD2171, pn-vegf receptor tyrosine kinse inhiitor, normlizes tumor vsculture nd llevites edem in gliolstom ptients. Cncer Cell 2007; 11: Dings RP, Loren M, Heun H et l. Scheduling of rdition with ngiogenesis inhiitors nginex nd Avstin improves therpeutic outcome vi vessel normliztion. Clin Cncer Res 2007; 13: Ning T, Jing M, Peng Q et l. Low-dose endosttin normlizes the structure nd function of tumor vsculture nd improves the delivery nd nti-tumor efficcy of cytotoxic drugs in lung cncer xenogrft murine model. Thorcic Cncer 2012; 3: Gordon S, Tylor PR. Monocyte nd mcrophge heterogeneity. Nt Rev Immunol 2005; 5: Pollrd JW. Trophic mcrophges in development nd disese. Nt Rev Immunol 2009; 9: Ding T, Xu J, Wng F et l. High tumor infiltrting mcrophge density predicts poor prognosis in ptients with primry heptocellulr crcinom fter resection. Hum Pthol 2009; 40: Lee CH, Espinos I, Vrijldenhoven S et l. Prognostic significnce of mcrophge infiltrtion in leiomyosrcoms. Clin Cncer Res 2008; 14: Thorcic Cncer 4 (2013) Tinjin Lung Cncer Institute nd Wiley Pulishing Asi Pty Ltd 303

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