Lentinan inhibits tumor angiogenesis via interferon γ and in a T cell independent manner

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1 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 RESEARCH Open Access Lentinn inhiits tumor ngiogenesis vi interferon γ nd in T cell independent mnner Shengming Deng 1, Guoxi Zhng 2, Jijie Kui 1, Peng Fn 1, Xuexing Wng 1, Pei Zhou 1, Dn Yng 3, Xichen Zheng 1, Xiomei Liu 1, Qunli Wu 3* nd Yuhui Hung 1* Astrct Bckground: Antingiogenic gents re commonly used in lung nd colon cncer tretments, however, rpid development of drug resistnce limits their efficcy. Methods: Lentinn (LNT) is iologiclly ctive compound extrcted from Lentinus edodes. The effects of LNT on tumor ngiogenesis were evluted y immunohistochemistry in murine LAP0297 lung nd CT26 colorectl tumor models. The impcts of LNT on immune cells nd gene expression in tumor tissues were determined y flow cytometry, qpcr, nd ELISA. Nude mice nd IFNγ lockde were used to investigte the mechnism of LNT ffecting on tumor ngiogenesis. The dt sets were compred using two-tiled student s t tests or ANOVA. Results: We found tht LNT inhiited tumor ngiogenesis nd the growth of lung nd colon cncers. LNT tretments elevted the expression of ngiosttic fctors such s IFNγ nd lso incresed tumor infiltrtion of IFNγ-expressing T cells nd myeloid cells. Interestingly, IFNγ lockde, ut not T cell deficiency, reversed the effects of LNT tretments on tumor lood vessels. Moreover, long-lsting LNT dministrtion persistently suppressed tumor ngiogenesis nd inhiited tumor growth. Conclusions: LNT inhiits tumor ngiogenesis y incresing IFNγ production nd in T cell-independent mnner. Our findings suggest tht LNT could e developed s new ntingiogenic gent for long-term tretment of lung nd colon cncers. Keywords: Lentinn, Interferon γ, Antingiogenic therpy, Drug resistnce, Lung cncer Bckground New lood vessels must e developed to nourish tumors tht grow igger thn 2 mm 3 in volume, nd the tumor vsculture is lso necessity for tumor s ility to metstsis; ngiogenesis is thus hllmrk of cncer [1, 2]. Importnt prongiogenic fctors tht re known to e involved in tumor ngiogenesis include proteins of the vsculr endothelil growth fctor (VEGF) fmily, the * Correspondence: chinlie@163.com; hungyh@sud.edu.cn Shengming Deng nd Guoxi Zhng contriuted eqully to this work. 3 Deprtment of Trditionl Chinese Medicine, Peking Union Medicl College Hospitl, Peking Union Medicl College nd Chinese Acdemy of Medicl Sciences, No. 1 Shuifuyun, Dongcheng District, Beijing , Chin 1 Cyrus Tng Hemtology Center, Collortive Innovtion Center of Hemtology, Stte Key Lortory of Rdition Medicine nd Prevention, Soochow University, 199 Ren-Ai Rod, Suzhou , Jingsu, Chin Full list of uthor informtion is ville t the end of the rticle pltelet-derived growth fctor (PDGF) fmily, the firolst growth fctor (FGF) fmily, nd plcentl growth fctor (PlGF) [1, 3]. Therefore, the lockde of prongiogenic signling pthwys hs een investigted nd developed s n ntingiogenic therpy iming to strve tumor cells to deth [3 6]. Antingiogenic therpies re currently in wide use ginst non-smll cell lung cncer (NSCLC), colorectl cncer, nd severl other types of solid tumors [1, 3]. The first ntingiogenic gent pproved for NSCLC ws evcizum, monoclonl ntiody to VEGF-A. The comintion of evcizum nd chemotherpeutic gents, such s cropltin nd pclitxel, improved oth progression-free survivl nd overll survivl in dvnced NSCLC ptients, compred with chemotherpy lone [4]. However, the clinicl enefits from VEGF inhiitors re modest nd trnsient, The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 2 of 12 ndreusullyfollowedytherpidemergenceofdrug resistnce [1, 3, 6]. As compenstory prongiogenic fctors, such s PDGF nd FGF, ply key roles in mediting the resistnce to VEGF signling lockde therpy, multiple trgeted kinse inhiitors (TKIs) could circumvent the common prolem of rpid drug resistnce onset ecuse they simultneously lock severl signling pthwys [1, 3]. The development of multitrgeted TKIs is expected to comt this resistnce, however, there hve een few convincingly studies in the clinic to dte [1, 7]. Moreover, mrginl survivl enefits nd significnt toxicities ssocited with TKIs hve limited enthusism for this therpeutic pproch. Therefore, new strtegy to etter control tumor ngiogenesis remins much nticipted. Severl recent reports suggest tht immune stimultion cn lso restrict tumor ngiogenesis. Activted T cells hve een shown to decrese tumor vessel density, inhiit tumor endothelil cell prolifertion, nd/or rrest tumor lood flow [8 12], suggesting immune stimultors might hve the cpility to inhiit tumor ngiogenesis. Lentinn (LNT), iologiclly ctive compound extrcted from Lentinus edodes (L. edodes), is n immunopotentitor [13 15], nd exhiits multiple iologicl ctivities, including immunomodultory, nticteril, ntivirus, ntitumor effects, nd nti-inflmmtory [14, 16 19]. Although the comintion of LNT with TNP-470 (TNP), n ngiogenic inhiitor, displyed ntingiogenic effects nd promoted tumor cell poptosis [20], whether LNT ffects tumor ngiogenesis remins uncler. In this study, we evluted the effects of LNT on the tumor vsculture in LAP0297 lung crcinom nd CT26 colorectl crcinom. LNT tretments significntly reduced tumor vsculr function nd inhiited tumor growth. Moreover, long-term LNT tretments continuously suppressed tumor ngiogenesis nd exhiited ntitumor effects. Mechniclly, LNT inhiited tumor ngiogenesis vi IFNγ up-regultion, which ws ssocited with the ccumultion of tumorinfiltrting myeloid cells. Thus, this study demonstrtes the potentil of LNT to e served s novel ntingiogenic gent for long-term cncer tretments. Methods Mterils nd regents The Lentinn (LNT, 1 mg, ), gift from Nnjing Luye phrmceuticl Co., Ltd. (Nnjing, Chin), is provided s powder in penicillin ottle. LNT ws dissolved in sline (0.9% NCl) right efore in vivo dministrtion. Tumor models Femle FVB mice (6 8 weeks old) were red nd mintined in the gnotoiotic lortory niml center in Soochow University. Femle BALB/c nd nude mice (6 8 weeks old) were purchsed from the Shnghi Lortory Animl Center (Shnghi, Chin) nd the Vitl River Lortories (Beijing, Chin), respectively. All of the mice were housed in the specific pthogen-free (SPF) condition. FVB nd Bl/c mice were sucutneous (s.c.) inoculted with cells of LAP0297 or CT26 on the right flnks, respectively. When tumors reched 4 5 mm in dimeter, mice ering tumors were rndomly divided into pproprite groups nd sujected to Lentinn or sline (0.9% NCl) tretments. In the long-term tretment experiments, mice ering tumors were euthnized when tumor volume exceeded 1000 mm 3. Tumor volume (mm 3 ) ws estimted y using the following formul: tumor volume = (long xis) (short xis) 2 π/6. Cell lines The LAP0297 lung crcinom cell line ws generted y Dr. Peigen Hung t Msschusetts Generl Hospitl (Boston, USA) [21]. The CT26 tumor cell line ws purchsed from the Americn Type Culture Collection. Tumor cells were cultured in Dulecco s modified Egle s medium (DMEM) (GIBCO) supplemented with 10% het-inctivted fetl ovine serum (FBS) (GIBCO) nd 1% penicillin nd streptomycin (GIBCO) t 37 C in humid incutor contining 5% CO 2. Cell cultures were frequently monitored for mycoplsm contmintion, nd only mycoplsm-negtive cells were used for experiments. Tumor vsculr function nlysis The function of tumor lood vessels ws determined y nlyzing the intensity of Hoechst (Sigm), s descried previously [22]. Briefly, 5 min fter intrvenous injection of Hoechst (10 mg/kg), mice were systemiclly perfused with PBS, nd the tumors were removed nd fixed with 4% prformldehyde. This procedure leled functionl vessels with fluorescence of nucleus-ound Hoechst Mosic imges of tumors were tken using n Olympus FV1000 confocl lser-scnning microscope. A 20 ojective cquired μm tiles, nd n utomted stge scnned through the entire cross section of tumor tissue. The imged tiles were stitched into finl mosic imge using n Olympus softwre. Nonspecific nucler stining (Sytox green from Moleculr Proes) ws used to counter-stin the slides. In ech field, the intensities of CD31 nd Hoechst positive res were clculted using n Imge-Pro plus softwre (version 6.0). Immunohistochemistry Tumor tissue smples were fixed for 2 3 h in 4% prformldehyde, nd then incuted with 30% sucrose in PBS overnight t 4 C. The tissue smples were then emedded in opticl coherence tomogrphy (OCT) compound nd stored t 80 C. Frozen sections (20 μm) were incuted with primry rt nti-mouse CD31 ntiody (endothelil cell mrker, 1:100, Clone MEC13.3, Ct#: , BD

3 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 3 of 12 Biosciences) nd secondry Alex Fluor 647 donkey nti-rt IgG ntiody (1:200, Jckson ImmunoReserch) to stin endothelil cells. The slides were counter-stined for cell nuclei y Sytox Green (Moleculr Proes). Fluorescent imges were tken using n Olympus FV1000 confocl lser scnning microscopy. Four to six photogrphic res, excluding the tumor periphery, were rndomly tken from ech tumor tissue ( μm 2 ech). Men fluorescence intensity (MFI) of CD31 positive nd Hoechst stined res were clculted using n Imge-Pro plus softwre (version 6.0). Tue formtion ssys Tue formtion ssy ws used to evlute the effect of LNT on endothelil cells s descried previously with minor modifictions [23, 24]. Briefly, humn umilicl vein endothelil cells (HUVECs) were cultured in DMEM medium contining 10% FBS. Growth fctor reduced mtrigel mtrix (CORNING) ws plced in refrigertor (4 C) overnight to thw the mtrigel. Pltes (24-well) were coted with 100 μl/well of mtrigel mtrix nd were incuted t 37 C for 30 min to llow the gel to solidify. HUVECs (30,000 cells/well) were seeded onto the top of the gel nd were treted with different concentrtions of LNT for 12 h in triplicte t 37 C with 5% CO 2.The process of tue formtion ws monitored every 3 h nd the pictures were tken y using Box-Type Fluorescence Imging Device (OLYMPUS). The numers of the tues nd rnches in ech well were counted. Quntittive rel-time PCR Totl RNA from tumor tissues ws isolted y MicroElute Totl RNA kit (Omeg), followed y cdna synthesis with RevertAid First Strnd cdna Synthesis Kit (Thermo Scientific). The levels of relted mrna were determined y using high-throughput fluorescence quntittive PCR meter (LightCycler480 II) (Roche). The primers (Tle 1) were specificlly designed to void nonspecific mplifiction y one hlf hyridizing to the 3 end of oneexonndtheotherhlfhyridizingtothe5 end of the djcent exon. Bet-ctin ws used s reference gene to clculte the differences in gene expression (fold chnge). Flow cytometry nlysis Mice ering tumors were intrcrdilly perfused with PBS. Tumor tissues were isolted nd single cell suspensions were prepred y using the digesting DMEM medium contining collgense type 1A (1500 U/ml), hyluronidse (1000 U/ml) nd DNse (20 U/ml). Rt nti-mouse CD16/CD32 monoclonl ntiody ws dded into the single-cell suspensions efore other ntiody stining. After stining, cells were wshed with cold flow uffer (1% BSA, 0.1% NN 3 in PBS), nd 7AAD regent (ebioscience) ws dded (5 ul/tue) prior to running the Tle 1 Primers used for qpcr nlysis Gene Primer Sequence (5-3 ) β-ctin Forwrd ATCGTGCGTGACATCAAAGA ACAGGATTCCATACCCAAGAAG Cxcl9 Ifnγ Tnfα Ang1 Tsp1 Timp1 Forwrd Forwrd Forwrd Forwrd Forwrd Forwrd AGTGTGGAGTTCGAGGAACC GAGTCCGGATCTAGGCAGG CCAAGTTTGAGGTCAACAACCC GGGACAATCTCTTCCCCACC CCGATGGGTTGTACCTTGTC CGGACTCCGCAAAGTCTAAG ATGGAAAATTATACTCAGTGGCTGC ATTTAGTACCTGGGTCTCAACATC TGTCACTGCCAGAACTCGGTTA GGAGACCAGCCATCGTCAG GAGACACACCAGAGCAGATACC GCTGGTATAAGGTGGTCTCGT flow nlysis. For intrcellulr cytokine stining, 2 million cells were cultured in 12-well plte for 4 h with Brefeldin A(10 μg/ml, ebiosciences), nd then stined with the surfce ntiody mixture. After wshed, intrcellulr stining ws performed ccording to the mnufcturer s instructions of Fixtion/Permeiliztion Solution Kit (BD Bioscience). Flow cytometry dt were cquired on Gllios flow cytometer (Beckmn) nd nlyzed with Kluz softwre (version 1.3). The following fluorescence-leled or isotype-mtched nti-mouse ntiodies were used: Ly-6G-FITC, CD8-FITC, CD4-PE, NK1.1-APC-Cy7, Ly- 6C-PE, Gr1-APC-Cy7, IFNγ-PE-Cy7, nd IFNγ-APC (BD Biosciences); F4/80-APC, CD45-BV421, nd CD11-BV 510 (BioLegend). In vivo IFNγ neutrliztion When LAP0297 lung tumors reched 4 5 mm in dimeter, mice ering tumors were rndomly divided into four groups, which were treted with n isotype-mtched control rt IgG1 (clone HRPN, Bio-X Cell), 1 mg/kg LNT, 250 μg nti-ifnγ ntiody (clone XMG1.2, Bio-X Cell), or LNT plus n nti-ifnγ ntiody. LNT ws dministered into mice vi i.p. injection every 3 dys, while rt IgG1 or nti-ifnγ ntiody were given every 3 dys. The tretment durtion ws 10 dys. Enzyme-linked immunosorent ssy (ELISA) The concentrtions of ngiosttic fctors IFNγ, TNFα, CXCL9, Ang1, TSP1, nd TIMP1 in the tumor tissue lyste were mesured ccording to the mnufcturer s protocols y using the following mouse ELISA Kits: IFNγ (Ct#: DKW , Dkewe, Shnghi, Chin), TNFα (Ct#: DKW , Dkewe, Shnghi, Chin), CXCL9 (Ct#: 70-EK21432/2, MultiSciences, Hngzhou, Chin), Ang1 (Ct#: DL-ngpt1-mu-96 t), TIMP1 (Ct#: DL-timp1-mu-96 t), nd TSP1 (Ct#: DL-ths1-mu-96 t) from DLDEVELOP (Wuxi, Chin). Although different kits

4 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 4 of 12 hve different mesure protocols, the mjor procedure is similr. Here, we used IFNγ mesurement s n exmple of the ELISA method. Briefly, IFNγ stndrds, lnk control nd tested smples (100 μl/well) were dded into 96-well pltes. Then Biotinylted ntiody (50 μl/well) ws dded to ech well nd incuted t 37 C for 90 mins. After wshing wy unound Biotinylted ntiody, Streptvidin- HRP (100 μl/well) ws dded to ech well nd incuted for 30 mins. After wshing, TMB(100 μl/well) ws dded nd incuted t 37 C for 10 mins in the drk. The rection ws discontinued y the Stop solution nd the opticl density (OD) vlues were determined t the wvelength of 450 nm y microplte reder. The concentrtions of ngiosttic fctors were clculted using the stndrd curve s regression eqution derived from stndrd sornce vlues. Sttisticl nlysis Sttisticl nlyses were performed using Prism sttisticl softwre (version 6, GrphPd Softwre, Inc.). Unpired 2-tiled Student s t tests were used to determine the sttisticl differences etween two groups. One-wy nlysis of vrince (ANOVA) ws used to ssess the differences when more thn two groups were compred. Dt were presented s men ± stndrd error of the men (SEM). The results were considered s sttisticlly significnt t P < 0.05 (*). P vlues lower thn 0.01 or were indicted s ** or ***, respectively. Results LNT tretments exhiit etter tumor growth inhiition t reltively low dosge Tumor immune evsion nd tumor ngiogenesis re two hllmrks of cncer [2, 22, 25]. LNT hs een shown to suppress the growth of severl types of cncer nd the effects hve een lrgely ttriuted to immune stimultion [13, 14, 16, 18, 26, 27]. Whether LNT intervention influences tumor ngiogenesis remins uncler. To determine the impct of LNT on tumor ngiogenesis, we initilly evluted the dose effects of LNT tretments on LAP0297 lung crcinom. We treted tumor-ering mice with 0.25, 0.5, 1.0, 2.0, or 5.0 mg/kg LNT for 10 dys (Fig. 1). All tested dosges of LNT tretments inhiited LAP0297 lung tumor growth, however, reltively lower doses exhiited etter tumor control thn higher doses, suggesting tht LNT tretment inhiited tumor growth in n inverted U-shped dose-response mnner. Our dt showed tht the 1.0 mg/kg LNT tretments exhiited etter ntitumor effects, compred to the 5.0 mg/kg LNT tretments (Fig. 1-c). Bsed on these results, we chose 1.0 mg/kg s the stndrd LNT tretment dose for LAP0297 lung tumors in the rest of this study. This unique dose effect of LNT ws lso oserved in the CT26 colorectl cncer model, where 1.0 mg/kg LNT tretments inhiited tumor growth, ut 4.0 mg/kg LNT tretments did not ffect tumor growth, gin showing tht lower dose of LNT tretment hs etter ntitumor effects (Additionl file 1: Figure S1). Tken together, these dt show tht ppropritely lower doses of LNT tretments exhiit etter ntitumor effects, compred to those of higher doses. LNT tretments reduce tumor vsculr function in lung cncer We next investigted the effects of LNT tretments on tumor ngiogenesis. Vessel density is common prmeter to evlute the effects of n intervention on the tumor vsculture [22]. After 10 dys of LNT tretments in the LAP0297 lung cncer model, vessel density in ll of LNT-treted groups ws comprle to the control group, even though tumors were smller in LNT-treted groups when compred to the control group (Figs. 1 nd 2). We then nlyzed other prmeters to get more informtion out the tumor vsculture. Interestingly, we found tht LNT tretments suppressed tumor vsculr function in n inverted U-shped dose-response mnner (Fig. 2). A reltively lower tretment dose of LNT (1.0 mg/kg) exhiited stronger ntingiogenic effects, compred to reltively higher dose (5.0 mg/kg) (Fig. 2). Given tht the tumor microenvironment is highly heterogeneous nd tumor vessels re unevenly distriuted [28, 29], we next looked t the whole imges of the entire cross-sections of tumor tissues nd nlyzed the overll tumor vsculr function. Agin, the dt showed tht LNT tretments (1.0 mg/kg) decresed tumor vsculr function (Fig. 3). To evlute whether LNT tretments ffect lood vessels in norml tissues, we exmined lood vessels in colon tissues nd found tht LNT tretments didn t lter their vessel density or function (Additionl file 1: Figure S2). Consistent to the reduction in tumor vsculr function, the necrotic re in the LNT-treted group ws incresed compred to the control group (Additionl file 1: Figure S3). Tken together, our dt suggest tht LNT tretments suppress tumor ngiogenesis in the lung cncer model. LNT tretments suppress tumor vsculr function in colorectl cncer To determine whether the ntingiogenic effects of LNT tretments re tumor-type dependent, we treted mice ering CT26 colorectl cncer with LNT. Consistent with the effects seen in the LAP0297 lung cncer model, 10 dys of LNT tretments (1.0 mg/kg) inhiited CT26 tumor growth (Additionl file 1: Figure S1). We then nlyzed the effects of LNT tretments on tumor ngiogenesis. LNT tretments (1.0 mg/kg) significntly decresed the Ho33342 perfused tumor re, ut did not chnge the density of tumor lood vessels (Fig. 4). The

5 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 5 of 12 c Fig. 1 Lentinn (LNT) exhiits etter ntitumor effects t reltively lower dose compred with higher dose. Experimentl design: FVB mice were sucutneously (s.c.) inocultedwith LAP0297 lung cncer cells on the right flnk. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 6 groups nd received i.p. injection of different doses of LNT (0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, nd 5.0 mg/kg) dily for 10 dys. LAP0297 lung tumor-ering mice received different doses of LNT tretments. c Tumor weight ws mesured t the end of the tretments. NS: control group treted with sline. All dt in this report re presented s mens ± SEM. * p <0.05,**p < 0,01, *** p <0.001 c Fig. 2 LNT tretments reduce tumor vessel function in n inversed U-shped dose-response mnner. LAP0297 tumor ering mice were treted with different doses of LNT for 10 dys s descried in Fig. 1. Tumor tissues were sectioned nd stined with n nti-cd31 ntiody. Representtive figures showing tumor vessel density nd vsculr function in NS control nd LNT (1 nd 5 mg/kg) groups. The men fluorescence intensity (MFI) of CD31 (Red) nd Hoechst (Blue) stined res ws quntified. CD31 is n endothelil cell mrker. The intensity of Hoechst perfusion reflects the function of tumor vessels. ns: no significntly different, * p < 0.05, *** p <0.001

6 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 6 of 12 Fig. 3 The whole imges of tumor tissue cross-sections show reduced tumor vsculr function fter LNT tretments in LAP0297 lung cncer. LAP0297 tumor ering mice received sline or LNT tretments (1.0 mg/kg) for 10 dys s descried in Fig. 1. Tumor tissues were sectioned nd stined with n nti-cd31 ntiody. The proportions of endothelil cell re (CD31, Red) nd Hoechst (Blue) stined re over totl tumor tissue re were quntified. CD31 is n endothelil cell mrker. The intensity of Hoechst perfusion reflects the function of tumor vessels. *** p < dt show tht LTN tretments inhiit colorectl tumor vsculr function, indicting tht LNT cn e used s n ntingiogenic gent for colorectl cncer tretment. Long-term LNT tretments induce the regression of lung cncer Antingiogenic therpy is widely used in the clinic nd shows modest ntitumor efficcy in severl cncer types, such s NSCLC nd colorectl cncer. One crucil chllenge for ntingiogenic therpy is the emergence of rpid drug resistnce [1, 6, 29]. To determine the long-term ntingiogenic effect of LNT tretment, we continuously treted mice ering LAP0297 lung cncer with LNT (1.0 mg/kg) for 1 month. During the LNT tretment course, the size of LAP0297 lung cncers persistently reduced nd out 60% of tumors were completely regressed t the end of the tretment course (Fig. 5). We kept monitoring the tumor growth when LNT tretment ws terminted, nd found tht there ws no oserved LAP0297 lung tumor regrowth 2 weeks post the LNT tretment course, nd the percentge of tumor free mice reched 95% (Fig. 5). These results suggest tht long-lsting LNT tretments exhiit persistent ntitumor effects in the LAP0297 lung cncer model. LNT tretments upregulte the expression of ngiosttic fctors To understnd the mechnism of LNT inhiition ginst tumor ngiogenesis, we performed endothelil cell tue formtion ssys, nd found tht LNT tretments did not directly ffect endothelil cell tue formtion (Additionl file 1: Figure S4). We then nlyzed the trnscription of ngiogenesis-relted genes, nd found tht ngiosttic genes, including interferon γ (Ifnγ), tumor necrosis fctor α (Tnfα), chemokine (C-X-C motif) lignd 9 (Cxcl9), Timp1, nd Thromospondin 1 (Tsp1), were up-regulted in LNT-treted tumors, compred to control tumors (Additionl file 1: Figure S5). Moreover, we conducted ELISA to determine the protein levels of the ngiosttic fctors in the tumor microenvironment. The results demonstrted tht LNT tretments (1 mg/kg) elevted the protein levels of IFNγ, TNFα, CXCL9, nd TIMP1 (Additionl file 1: Figure S6). These dt collectively suggest tht LNT tretments induce the expression of ngiosttic fctors. LNT tretments increse IFNγ production in T cells nd myeloid cells Since IFNγ is often secreted y immune cell popultions upon stimultion, we next nlyzed the effects of LNT tretments on tumor-infiltrting immune cells. Flow cytometric nlysis showed tht LNT tretments fcilitted tumor infiltrtion of CD4+, CD8+, NK1.1+ cells, monocytes, nd neutrophils, while reducing tumor-ssocited mcrophges (TAMs) (Fig. 6). Intrcellulr stining further showed tht LNT tretments incresed the frctions of IFNγ-expressing T cells nd NK cells, s well s IFNγ-expressing monocytes nd neutrophils, ut not TAMs (Fig. 6). The dt suggest tht LNT tretments promote the ccumultion of IFNγ-expressing T cells nd myeloid cells.

7 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 7 of 12 c Fig. 4 LNT tretments significntly reduce tumor vsculr function in CT26 colorectl crcinom. CT26 colorectl crcinom cells ( ) were s.c. injected on the right flnk of BALB/c mice. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 2 groups nd received i.p. injection of sline or 1.0 mg/kg of LNT for 10 dys. Tumor vsculr prmeters were determined s descried in Fig. 2. Representtive figures showed tumor vessel density nd vsculr function. The quntifictions of MFI of Hoechst33342 perfused tumor res in sline (NS) or LNT treted tumors. c The MFI of tumor vessel density in sline or LNT treted tumors. ** p <0.01 IFNγ is required for LNT tretment to reduce tumor vsculr function Previous studies hve suggested tht IFNγ-expressing T cells could suppress tumor ngiogenesis [8, 12]. Together with the reports tht LNT is commonly used s n immune modultor, nd tht T cells re often mjor ntitumor immune effector cells [13, 14, 18, 28], we thus tested whether LNT exerts its ntingiogenic effects through T cells. We inoculted LAP0297 lung cncer cells in nude mice nd then treted mice with 1.0 mg/kg LNT for 10 dys s did in immune competent mice (Fig. 1). We found tht LNT tretments inhiited the growth of LAP0297 lung cncer in nude mice (Additionl file 1: Figure S7), ut the degree of inhiition ws less thn tht in immune competent mice (Fig. 1), indicting tht T cells contriute to the inhiition of tumor growth y LNT tretments. This is consistent with the immunopotentition of LNT. We lso nlyzed tumor vessels, nd found tht LNT tretments suppressed tumor vsculr function in nude mice (Fig. 7), nd tht the degree of suppression ws comprle to those of immune competent mice (Fig. 2). LNT tretments lso slightly reduced tumor vessel density (Fig. 7), which ws not oserved in immune competent mice (Fig. 2). These dt suggest tht LNT tretments inhiit tumor ngiogenesis in T cell-independent mnner. A recent study demonstrted tht elevted levels of IFNγ rrested tumor lood flow without ffecting tumor endothelil cell prolifertion nd poptosis [10]. To further explore the reltionship etween IFNγ nd LNT tretment, we tested whether IFNγ is responsile for the oserved LNT-medited suppression of vsculr function. In vivo neutrliztion of IFNγ prtilly reversed the inhiition of tumor growth y LNT tretments compred with the control group, while IFNγ neutrliztion lone hd little effect on tumor growth (Fig. 8-). Consistent with previous dt, LNT tretments lone reduced tumor vsculr function, nd IFNγ neutrliztion negted the effect of LNT tretments on tumor vsculr function, indicting tht IFNγ medites the impct of LNT tretments on tumor vsculr function (Fig. 8). Tken together, these dt suggest tht LNT tretments

8 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 8 of 12 c Fig. 5 Long-term LNT tretments induce regression of LAP0297 lung cncer. LAP0297 tumor ering mice were prepred s descried in Fig. 1. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 2 groups nd received i.p. injection of sline or LNT (1.0 mg/kg) for 1 month. Representtive photogrphs of LAP0297 lung cncer-ering mice in sline nd LNT-treted group were tken t the end of the experiment. The growth curves of LAP0297 lung cncer upon long-term therpy of sline or LNT. c The percentge of tumor free mice on dy 45 fter tumor inocultion (NS group, n = 17 mice, LNT-treted group, n = 24 mice). *** p < suppress tumor ngiogenesis vi IFNγ nd in T cell-independent mnner, which is correlted with n increse in IFNγ-expressing myeloid cells. Discussion Tumor initition nd progression relies on the formtion of new lood vessels. Thus, the disruption of tumor lood vessels hs the potentil to inhiit tumor growth. However, the clinicl enefits of ntingiogenic therpies re often in the order of weeks nd rpidly developed drug resistnce limits its long-term ppliction [1, 6, 29]. In this study, we found tht oth of T cells nd IFNγ production contriute to tumor growth inhiition induced y LNT tretments, wheres IFNγ, ut not T cells, Fig. 6 LNT tretments promote tumor infiltrtion of IFNγ-expressing T cells nd myeloid cells. LAP0297 tumor ering mice were prepred nd then treted with sline or 1.0 mg/kg LNT for 10 dys s descried in Fig. 1. Tumor tissues were isolted nd single cell suspensions were prepred for flow nlysis. Tumor infiltrtion of T cells nd myeloid cells. The proportions of IFNγ-expressing T cells nd myeloid cells in LAP0297 tumor tissues upon sline or LNT tretments. * p < 0.05, ** p < 0,01, *** p < 0.001

9 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 9 of 12 c Fig. 7 LNT tretments reduce tumor vsculr function in LAP0297 lung cncer in nude mice. Nude mice were s.c. inoculted with LAP0297 lung cncer cells on the right flnk. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 2 groups nd received i.p. injection of sline or 1.0 mg/kg of LNT for 10 dys. Tumor vsculr prmeters were determined s descried in Fig. 2. Representtive figures showing tumor vessel density nd function. The MFI of Hoechst33342 perfused tumor res nd the MFI of tumor vessel density c in sline or LNT treted tumors. ** p < 0.01, *** p <0.001 is required for the LNT-medited ntingiogenic effect. Moreover, prolonged LNT tretments persistently suppressed tumor ngiogenesis nd reduced tumor volume. These results suggest tht LNT could e served s new ntingiogenic gent nd my e suitle for long-term intervention. LNT is polyscchride from the fruit ody of L. edodes nd hs een used previously s iologicl response modifier [14, 15, 30]. In prticulr, LNT hs een pproved s n djuvnt for the tretment of gstric cncer nd rought clinicl enefits to cncer ptients [16, 31, 32]. LNT hs demonstrted its ntitumor effects in oth primry nd trnsplnted tumor models with negligile side effects [17, 33, 34]. Our dt further suggest tht LNT tretments decrese tumor vsculr function, ut do not ffect vsculr function in norml tissues. Such difference effects could e due to the fct tht LNT tretments suppress tumor ngiogenesis vi incresing IFNγ production, which is ssocited with the ccumultion of IFNγ-expressing neutrophils. Norml tissues usully contin few neutrophils nd will show miniml effects y LNT tretments. In ddition, LNT could llevite side toxicities of chemotherpeutic gents nd potentilly improve their efficcy [26, 35]. The sfety profiles of LNT s well s its ility to overcome the side effects of chemotherpy is superior to currently used trditionl ntingiogenic drugs, providing new rtionles for developing LNT s n ntingiogenic gent. Blood vessel formtion is tightly regulted y pro- nd nti-ngiogenic fctors. The relentless production of pro-ngiogenic fctors promotes tumor vessel formtion [3, 29]. Thus ntingiogenic therpy is usully designed to suppress pro-ngiogenic fctors nd inhiit tumor progression [1, 3]. Currently, ntingiogenic gents re used in the tretments of vrious types of solid cncers, such s NSCLC nd colorectl cncer [1, 3]. Therefore, we chose LAP0297 lung crcinom nd CT26 colorectl cncer s tumor models to evlute the effects of LNT tretments on tumor ngiogenesis. Although ntingiogenic therpy improves chemotherpy in severl cncer types, the clinicl enefits re mrginl. One of the criticl resons is the development of drug resistnce. Some tumors re intrinsic resistnce to ntingiogenic therpy [3, 29]. Some tumors respond to ntingiogenic therpy

10 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 10 of 12 c d Fig. 8 Anti-IFN-γ ntiody tretments reverse the effects of LNT tretments on tumor vessels in LAP0297 lung cncer. LAP0297 tumor ering mice were prepred s descried in Fig. 1. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 4 groups nd received i.p. injection of HRPN, LNT (1.0 mg/kg), nti-ifn-γ ntiody (250 μg/mouse) or LNT plus nti-ifn-γ ntiody for 10 dys. Tumor vsculr prmeters were determined s descried in Fig. 2. nd The growth curves nd weight of LAP0297 tumors treted with HRPN, LNT, nti-ifn-γ ntiody, or the comintion of LNT nd nti-ifn-γ ntiody. c Representtive figures showing tumor vsculr function nd vessel density. d The quntifictions of Hoechst33342 perfused tumor res nd tumor vessel density. ns: no significntly different, * p < 0.05, ** p < 0.01, *** p < t the eginning, ut develop drug resistnce lter on [1]. With ntingiogenic tretments eing susceptile to the development of drug resistnce, it is significnt tht we treted LAP0297 lung crcinom with LNT for 1 month nd did not oserved drug resistnce. Gene trnscription nd protein expression dt showed tht LNT tretments upregulted the levels of IFNγ, TNFα, CXCL9, nd TIMP1. Among them, TIMP1 is n intrinsic ngiosttic fctor, while IFNγ, TNFα, nd CXCL9 re immune effector molecules with potent ngiosttic ctivities. Whether or not elevted endogenous ngiosttic fctors will e less likely to cuse drug resistnce is not known, ut the phenomenon is very interesting nd worth further investigtions. Therpeutic ntitumor drugs often suppress tumor growth in dose-dependent mnner. In generl, higher dosges show etter ntitumor effect thn tht of lower dosges. The optiml therpeutic dosge is usully determined y the efficcy nd toxicity of the drug. Interestingly, LNT inhiited tumor growth in n inversed U-shped dose-response mnner. Appropritely lower dose of LNT (such s 1.0 mg/kg) showed etter ntitumor efficcy thn tht of higher dose (such s 5.0 mg/kg). The exct underlying mechnism of this ction is unknown. It could e due to the nti-ngiogenic effects of LNT tretments. Our dt showed tht reltively lower dose of LNT tretments (such s 1.0 mg/kg) more potently reduced tumor vsculr function thn tht of reltively higher dose (such s 5.0 mg/kg). Furthermore, our study suggests tht LNT tretments (1.0 mg/kg) inhiit tumor vsculr function vi IFNγ production nd in T cell-independent mnner. In ddition, LNT tretments (1.0 mg/kg) up-regulted IFNγ production in severl tumor-infiltrting myeloid cell popultions. It is possile tht the optiml tretment dose of LNT could e different for different popultions of immune cells. The optiml tretment dose of LNT on specific popultion of immune cells with respect to their quntity nd function could e lso different. Indeed, the 1.0 mg/kg tretment of LNT, ut not the 5.0 mg/kg tretment of LNT, incresed tumor infiltrtion of neutrophils (Additionl file 1: Figure S8), which is the most undnt tumor-infiltrting myeloid cell popultion in LAP0297 lung crcinom (Fig. 6). Therefore, the optiml ntitumor effects of LNT tretments seem to depend on the lnce of vessel modultion nd immune stimultion upon LNT tretments. Since LNT tretment inhiits tumor growth in n inversed U-shped dose-response mnner, however, the lower or higher dose of LNT is reltive nd could e difficult to determine. This is n even igger chllenge in

11 Deng et l. Journl of Experimentl & Clinicl Cncer Reserch (2018) 37:260 Pge 11 of 12 the clinic ecuse different ptients nd different cncer types my hve different sensitivities to LNT tretments. Our previous work suggests tht monitoring vsculr function could e used to predict the efficcy of immune checkpoint therpy [36]. In this study, our findings showed tht reltively lower dose of LNT tretments ws more powerful thn higher dose in decresing tumor vessel perfusion. Given tht rdiologicl methods hve een developed to noninvsively mesure vessel perfusion during nti-ngiogenic therpy in the clinic [37, 38], it is conceivle tht vessel perfusion monitoring could e dpted to determine the optiml dosge of LNT tretment. LNT hs een pplied in some kinds of cncer tretments. Its ntitumor effects re usully considered to e the result of its immune stimultion. Severl recent studies suggested tht LNT could induce tumor cell poptosis, showing directly ntitumor effect [30, 39]. By using lung nd colorectl cncer models, we showed tht LNT tretments inhiited tumor ngiogenesis vi incresed IFNγ production in T cell-independent mnner. Tken together, LNT could ffect tumor growth vi multiple different mechnisms, including the modultion of immune system, the induction of tumor cell poptosis [13, 14, 16, 18, 30, 39], nd the suppression of tumor ngiogenesis. Conclusions LNT tretments reduced tumor vsculr perfusion nd inhiited tumor growth in n inverted U-shped dose-response mnner. Furthermore, long-term LNT tretments continuously suppressed tumor ngiogenesis nd exhiited ntitumor effects in LAP0297 lung tumor model. Mechniclly, LNT decresed tumor vsculr function y incresing IFNγ production nd in T cell-independent mnner, which ws ssocited with the ccumultion of tumor-infiltrting myeloid cells. Thus, this study suggests tht LNT could e served s new ntingiogenic gent for long-term cncer tretments. Additionl file Additionl file 1: Methods: CT26 colorectl crcinom cells (4x10 5 )or LAP0297 lung crcinom cells (2x10 5 ) were s.c. injected on the right flnk of BALB/c or FVB mice, respectively. When tumors reched 4 4 mm in dimeter, mice were rndomly divided into 2 groups nd received i.p. injection of sline (NS) or 1.0 mg/kg of LNT dily for 10 dys. At the end of the tretments, tumor tissues were isolted, sectioned, nd stined y n nti-cd31 ntiody (endothelil cells). The proportions of endothelil cell re (CD31, Red) nd Hoechst (Blue) perfused re over totl tumor tissue re were quntified. SYTOX Green (Green) ws used to lel totl cells. All dt in the Additionl file 1 re presented s mens ± SEM. * p<0.05, ** p<0.01, *** p<0.001, nd ns designtes no significnt difference. Fig. S1 LNT tretments inhiit tumor growth of CT26 colorectl crcinom. The experimentl design of CT26 colorectl tumor model. The growth curves nd weight of CT26 colon tumors treted with sline or LNT. Fig. S2 LNT tretments do not ffect the vsculture in norml colon tissues in LAP0297 tumor-ering mice. Fig. S3 LNT tretments increse necrotic re. LAP0297 tumor tissues were sectioned nd SYTOX Green (Green) ws used to lel tumor tissue cells. Necrotic cells were reltively smll, dense, nd round. Fig. S4 LNT tretments do not ffect endothelil cell tue formtion in vitro. HUVECs were seeded on the top of growth fctor reduced mtrigel in 24-well plte, nd then incuted with different concentrtions of LNT. The process of tue formtion ws monitored every 3 hrs nd pictures were tken t 12 hrs fter incution with LNT. The tues nd rnches in ech well were counted. Fig. S5 LNT tretments upregulte the trnscription of ngiosttic fctors, such s Ifnγ, Tnfα, Cxcl9, Ang1, Timp1,ndTsp1, in LAP0297 tumor tissues. Fig. S6 LNT tretments increse the expression of ngiosttic fctors, such s IFNγ,TNFα,CXCL9,Ang1,TIMP1, nd TSP1, in LAP0297 tumor tissues. Fig. S7 LNT tretments inhiit tumor growth of LAP0297 lung cncer in nude mice. Nude mice were s.c. injected with LAP0297 lung cncer cells on the right flnk nd tretments were performed s descried in the ove methods. Tumor sizes were mesured every three dys. Fig. S8 Reltively lower dose of LNT tretments (1 mg/kg), ut not higher dosge (5 mg/kg), promotes the tumorl ccumultion of neutrophils. LAP0297 tumor tissues were isolted nd single cell suspensions were prepred. The tumor infiltrtion of leukocytes (CD45), neutrophils (Gr1 hi ), nd TAMs were determined y flow nlysis. (ZIP 7212 k) Arevitions CXCL9: Chemokine (C-X-C motif) lignd 9; FGF: Firolst growth fctor; IFNγ: Interferon gmm; LNT: Lentinn; NSCLC: Non-smll cell lung cncer; PDGF: Pltelet-derived growth fctor; PlGF: Plcentl growth fctor; TIMP1: TIMP metllopeptidse inhiitor 1; TKI: Multiple trgeted kinse inhiitors; TNFα: Tumor necrosis fctor lph; TSP1: Thromospondin 1; VEGF: Vsculr endothelil growth fctor Acknowledgments The uthors thnk Dr. Wen Jing of MD Anderson Cncer Center for criticl reding nd comments. Funding This work ws supported in prt y grnts from the Ntionl Nturl Science Foundtion of Chin ( , ), the fund of the Distinguished Professors of Jingsu Province (SR ), the Collortive Innovtion Center of Hemtology, nd the Priority Acdemic Progrm Development of Jingsu Higher Eduction Institutions. Avilility of dt nd mterils All dt generted or nlyzed during this study re included in this pulished rticle. Authors contriutions YH, QW, nd GZ designed experiments, nlyzed dt nd wrote the mnuscript; SD, JK, PF, XW, ZP, DY, XZ, nd XL performed experiments nd nlyzed dt. QW nd YH supervised the study. All uthors red nd pproved the finl mnuscript. Ethics pprovl All niml works were pproved y the Institutionl Lortory Animl Cre nd Use Committee of Soochow University. All of the procedures were performed in complince with the Animl Cre nd Use Regultions of Chin. Consent for puliction Not pplicle. Competing interests The uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Cyrus Tng Hemtology Center, Collortive Innovtion Center of Hemtology, Stte Key Lortory of Rdition Medicine nd Prevention, Soochow University, 199 Ren-Ai Rod, Suzhou , Jingsu, Chin. 2 Nnjing Luye Phrmceuticl Co., Ltd, Nnjing , Jingsu, Chin.

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