Cancer Therapy: Preclinical Clinical Cancer Research Orally ... · previously (34). Cultured BMDCs at day 7 were used for experiments. BMDCs were further purified with anti-CD11c

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Published OnlineFirst September 20, 2010; DOI: 10.1158/1078-0432.CCR-10-0820

Clinical
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cer Therapy: Preclinical

lly Administered Particulate β-Glucan Modulatesor-Capturing Dendritic Cells and Improves

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itumor T-Cell Responses in Cancer

i1, Yihua Cai1, Chunjian Qi1, Richard Hansen1, Chuanlin Ding1, Thomas C. Mitchell2, and Jun Yan1

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pose: The beneficial properties of β-glucans have been recognized for centuries. Their proposednisms of action in cancer therapy occur via stimulation of macrophages and priming of innatephil complement receptor 3 for eliciting complement receptor 3–dependent cellular cytotoxicityb-opsonized tumor cells. The current study is to investigate whether β-glucan therapy has any effecttitumor adaptive T-cell responses.erimental Design: We first examined the trafficking of orally administered particulate yeast-d β-glucan and its interaction with dendritic cells (DC) that captured tumor materials. Antigen-ic T cells were adoptively transferred into recipient mice to determine whether oral β-glucan therapyes augmented T-cell responses. Lewis lung carcinoma and RAM-S lymphoma models were used toal β-glucan therapeutic effect. Further mechanistic studies including tumor-infiltrating T cells andne profiles within the tumor milieu were determined.ults:Orally administered particulate β-glucan trafficked into spleen and lymph nodes and activatedhat captured dying tumor cells in vivo, leading to the expansion and activation of antigen-specificnd CD8 T cells. In addition, IFN-γ production of tumor-infiltrating T cells and CTL responses werecantly enhanced on β-glucan treatment, which ultimately resulted in significantly reduced tumorn. Moreover, β-glucan–treated tumors had significantly more DC infiltration with the activated phe-e and significant levels of Th1-biased cytokines within the tumor microenvironment.clusions: These data highlight the ability of yeast-derived β-glucan to bridge innate and adaptive
Con
antitumor immunity and suggest that it can be used as an adjuvant for tumor immunotherapy. Clin Cancer

Res; 16(21); 5153–64. ©2010 AACR.

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logical response modifiers (BRM) derived frombial products have represented important tools forng mechanisms of host defense, but most BRMsremained classified as nonspecific because theirmode of action was unknown. β-Glucan BRMsirst reported 45 years ago and have been extensivelyigated for both their antitumor and anti-infectivey (1–4). Most β-glucan BRMs are derived from yeast,

, or fungi and have a backbone structure ofked D-glucose molecules (β-1,3-D-glucan).

tin-likfor sucells (showderiveantitumurincarcinreaffirligancriticaIn adimmuimmuopson

ns: 1Tumor Immunobiology Program of the Jamesncer Center, Department of Medicine and 2Instituteutics, Department of Microbiology and Immunology,ville School of Medicine, Louisville, Kentucky

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

uthor: Jun Yan, Tumor Immunobiology Program,rown Cancer Center, Clinical and Translationalg, Room 319, University of Louisville, 505 SouthLouisville, KY 40202. Phone: 502-852-3628; Fax:ail: [email protected].

0432.CCR-10-0820

ssociation for Cancer Research.

ls.org

Research. on November 9, clincancerres.aacrjournals.org d from

also have β-1,6–linked side chains of β1,3-D-glucanying sizes that occur at different intervals along theone (5). There are at least four β-glucan receptors thatbeen identified: complement receptor 3 (CR3;b/CD18, Mac-1, αMβ2 integrin; ref. 6), lactosylcera-(7), scavenger receptor (8), and dectin-1 (9–11). Inevious studies, we showed that lowmolecular weight,le β-glucan derived from yeast could bind to CR3 lec-e domain with high affinity and prime the receptorbsequent cytotoxic activation of iC3b-coated tumor12, 13). Furthermore, we and other investigators haven the therapeutic efficacy using combined yeast-d or barley β-glucan with complement activatingmor monoclonal antibodies (mAb) in a variety ofe syngeneic tumor models (14–17) as well as humanoma xenograft models (18–23). These studiesm that dual occupancy of CR3 by lectin-like domaind β-glucan and inserted domain ligand iC3b isl for β-glucan–mediated tumor immunotherapy.dition, yeast-derived β-glucan–mediated tumornotherapy uses a novel mechanism by which innate
ne effector neutrophils are primed to kill iC3b-ized tumor cells (16).
5153

2020. © 2010 American Association for Cancer

http://clincancerres.aacrjournals.org/

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Translational Relevance

Combined yeast-derived β-glucan with antitumormonoclonal antibody therapy is currently approvedby the Food and Drug Administration for its clinicalinvestigation in Phase I/II trials. The mechanism of ac-tion of β-glucan in cancer therapy occurs mainly viapriming innate effector neutrophil complement recep-tor 3 to induce cytotoxicity of iC3b-opsonized tumorcells. The current study showed that yeast-derived par-ticulate β-glucan exhibited potent adjuvant effect onpromoting antitumor T-cell responses. The findingswill not only advance our understanding of the actionmode of β-glucan but also have potential for directclinical utilization by enhancing the effect of cancerimmunotherapy.

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ereas these studies are mainly focused on the role ofans on innate immune cells, recent studies suggest-glucans may also possess the regulatory propertiesaptive immune responses. For example, bacterialan curdlan stimulates both Th1 and Th17 T-cell re-es (24) and exhibits a potent adjuvant effect on CD8priming (25). In addition, curdlan is capable of con-g regulatory T cells into Th17 cells using an in vitroe system (26). Th17 cells have been shown to play al role in antitumor T-cell responses (27–29). In con-ymosan β-glucan predominantly stimulates produc-f anti-inflammatory cytokines such as interleukin-10) and transforming growth factor-β (30). Zymosantreatment induces regulatory APCs and Ag-specifictolerance. In addition, β-glucan from Candidas stimulates human monocyte differentiation intoitic cells (DC), but these DCs inefficiently polarizeT cells (31). These new emerging data are of greatrtance and suggest a potential regulatory role ofans in eliciting adaptive T-cell responses. Thus, itcial to determine whether yeast-derived β-glucanent in cancer would promote or inhibit antitumorresponses.his study, we evaluated the potential effect of orallyistered particulate yeast-derived β-glucan as wholecan particles (WGP) on adaptive T-cell responses.rlier showed that oral WGPs were taken up byphages that transported them to spleen, lymphand bone marrow (BM). Within the marrow, thephages processed particulate β-glucan to releasesoluble active β-glucan moiety that bound to CR3trophils for eliciting cytotoxicity of iC3b-opsonizedcells (32). Here, we report that WGPs activate DCsapture tumor materials and that this subsequentlyto augmented Ag-specific CD4 and CD8 T-cell re-es. Orally administered WGP treatment caused sig-
t tumor regression with enhanced antitumor T-cell
ty and alteration of the tumor microenvironmentweretotal,

ancer Res; 16(21) November 1, 2010

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d Th1 responses. Therefore, particulate yeast-derivedcan links both innate and adaptive immune re-es and could serve as a potent adjuvant for effectiver immunotherapy.

rials and Methods

ntshly purified, soluble yeast β-glucan (PGG-Glucan)articulate β-glucan WGP isolated from the cell wallcharomyces cerevisiae were provided by Biothera. Thean preparation contained <0.02% protein, <0.01%an, and 1% glucosamine. For some studies, WGPsabeled with fluorescein dichlorotriazine (DTAF; Mo-r Probes-Invitrogen) as described previously (32).ndotoxin level was 0.06 EU/mL as tested by the gelethod (Associates of Cape Cod). Fluorochrome-gated antimouse mAbs including CD3 (17A2),(GK1.5), CD8 (53-6.7), CD11c (N418), Gr-1-8C5), CD25 (PC61), CD40 (HM40-3), CD800A1), CD86 (GL1), CD49b/pan-NK (DX5α), F4/80), MHC II (M5/114.15.2), and mouse Fc block2) were purchased from eBiosciences.

umin-transfected Lewis lung carcinoma cellsis lung carcinoma (LLC) cells were obtained frommerican Type Culture Collection. LLC cells were in-with pMiT-ovalbumin (OVA) retrovirus in the pres-f 10 μg/mL polybrene after 24 hours pretreatment250 ng tunicamycin (T7765; Sigma-Aldrich). LLChat stably expressed high levels of OVA were sortedoFlo High Speed Cell Sorter (Dako-Cytomation). Toe tumorigenicity, an additional selection was madevivo passage of the transfected cells in C57Bl/6 mice.umor cell lines with high OVA expression levelsapable of generating s.c. tumors were used forments.

and tumor modelsd-type C57BL/6 mice were purchased from Nationalr Institute. CD4 and CD8 OVA TCR transgenic (Tg)and OT-II mice were purchased from Taconic. Thee tumor protocols were done in compliance withevant laws and institutional guidelines and were ap-d by the Institutional Animal Care and Use Commit-University of Louisville.RMA-S-MUC1 tumor model, groups of C57Bl/6

received oral WGPs (100 μg, 200 μg, 400 μg, daily),ning on day 11 after palpable tumors were formed.were treated for 3 weeks with tumor diameter mea-ents made with calipers every third day, and miceacrificed when tumors reached 12 mm in diameter.were observed for tumor-free survival over 100For LLC/OVA tumor model, therapy was initiateds before mice were implanted s.c. with LLC/OVA1 × 107 per mouse). Two groups of C57Bl/6 mice
treated with 200 μL of WGPs (4 mg/mL in saline;800 μg) or 200 μL of PBS given every other day
Clinical Cancer Research



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an intragastric gavage needle. Therapy was contin-y administered for 3 weeks after palpable tumorsformed. Tumor diameters were measured everyday, and mice were sacrificed when tumors reachedm in diameter.

tion of tumor cell apoptosis and in vivory of dying tumor cells/OVA cells were cultured with 5 μmol/L staurospor-igma) for 6 hours to induce apoptosis. Apoptoticr cells were assessed by Annexin V-FITC apoptosision kit (BD Biosciences). Cells were labeled withxyfluorescein diacetate succinimidyl ester (CFSE;ular Probes) or PKH26 (Sigma) as described previ-(33). For in vivo delivery of dying tumor cells, 2 ×optotic CFSE-labeled LLC/OVA cells were injectedto mice. Mice were sacrificed 2 hours followingtumor cell injection, and the splenocytes wered for CD11c and CD8. In some experiments, mice re-g dying PKH26-labeled LLC/OVA cells were treatedr without orally administered WGPs (400 μg daily),he splenocytes were prepared and stained withc-allophycocyanin and FITC-conjugated mAbs, CD80, and CD86 and MHC class II or isotypel mAbs.

erived DCs-derived DCs (BMDC) were generated as describedusly (34). Cultured BMDCs at day 7 were used foriments. BMDCs were further purified with anti-c microbeads (Miltenyi Biotec, Inc.). Purity wasassessed by flow cytometry.

ro and in vivo T-cell proliferation assayin vitro assay, OT-I (CD8+) or OT-II (CD4+) T cellspurified by microbead separation (Miltenyi) fromcytes of OT-I or OT-II Tg mice. T cells (1 × 105) wereured with irradiated (2,500 rads) DCs (2 × 104) andLLC/OVA tumor cells (1 × 105) in the presence orce of WGP or PGG treatment for 72 hours. [3H]thy-e (1.0 μCi/well; ICN) was added during the last 16of culture. For in vivo assay, OT-I or OT-II T cellslabeled with 10 μmol/L CFSE for 10 minutes atas previously described (35). T cells (1 × 106 pere) were adoptively transferred into mice treated withhout oral WGPs. One day later, mice were injectedpoptotic LLC/OVA cells. Recipient mice were contin-y treated with WGPs for 5 days, and the turnoverells was examined by flow cytometry.

o cytotoxicity assaypreparation of target cells, splenocytes fromC57BL/6 mice were labeled with CFSE at 5 μmol/Lhigh) or 0.5 μmol/L (CFSElow). The CSFEhigh popula-as pulsed with 10 μg/mL of OVA SIINFEKL peptideours. Equal numbers of OVA peptide-pulsed CSFEhigh

o-peptide-pulsed CSFElow cells were mixed and adop-transferred into LLC/OVA tumor-bearing mice treated

differecomp

acrjournals.org


r without WGPs. Naive mice without tumors weres controls. After 24 hours, spleen cells were harvested,illing activity was assessed using flow cytometry.ntage killing was determined by CFSE+ gate afteralizing each sample to controls by the formularatioexperimental/ratiocontrol) × 100] where ratio = %igh/% CSFElow.

titative real-time PCRor samples were treated with TRIzol reagent

rogen), and total RNA was isolated and reverse-ribed with TaqMan reverse transcription reagentsied Biosystems). The indicated cytokine mRNA levelsquantified by quantitative real-time PCR amplifica-sing the BIO-RADMyiQ single color reverse transcrip-CR detection system. Briefly, cDNA was amplified inL reaction mixture containing 12.5 μL of SYBR Greenupermix (BIO-RAD), 100 ng of cDNA template, anded primers (200 nmol/L) using the recommendedg conditions (denaturation at 95°C for 10 minutesed by 40 cycles of 95°C for 15 seconds and 63°Cinute). The primer sequences, designed with Primer

ss software (Applied Biosystems), were summarizedplementary Table S1.

nofluorescence and immunohistochemistryngBL/6 mice were orally gavaged daily for 8 days withDTAF-WGPs and then sacrificed. Fresh spleen andnal lymph nodes were embedded in Tissue-Tekal cutting temperature compound 4853 (Electronscopy Science). Cryosections (7 μm) were fixed inld acetone and air-dried. Sections were stained withiotinylated rat anti-mouse CD11c (eBioscience) atvernight. After three washes with PBS, sections wereated with streptavidin-Alexa 594 (Molecular Probes)en imaged on a Leica TCS SP5 confocal microscopewith an HC PL APO 20×/0,7 CS (air) objective

Microsystems, Inc.).tumor sample immunohistochemistry staining, tu-ections were first blocked with avidin/biotin block-it (Vector Laboratories) and then stained withotinylated rat anti-mouse CD11c mAbs. After threees with PBS, sections were incubated with theavidin-horseradish peroxidase secondary antibodyo Scientific). Slides were rinsed in PBS and then

ated with the 3-amino-9-ethylcarbazole (AEC) sub-solution (Vector Laboratories). Slides were thenerstained with hematoxylin and mounted with-Mount mounting medium (Electron Microscopye).

ing and statistical analysis of dataa from each experiment were entered into Prism 4.0hPad Software) to generate bar graphs or graphs ofr regression and to determine the significance of
nces between data sets. Student's t test was used toare differences between each data set.
Clin Cancer Res; 16(21) November 1, 2010 5155



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lts

administered particulate β-glucan WGPsly interact with DCs in situ and stimulate DCshagocytose apoptotic tumor cells for maturationious studies have shown that orally administeredulate β-glucan WGPs are captured predominantlycrophages or mucosal M cells that transport themBM, spleen, and lymph nodes (32). To determineer oral WGP would directly interact with DCs,were fed orally with DTAF-labeled WGPs. Fluores-microscopy revealed that WGPs were readily seenth spleen and lymph nodes (Fig. 1A). Colocaliza-tudy showed that WGPs directly interacted withc+ DCs and some of the WGP particles werecytosed by DCs.next determined whether DCs could take up the ap-c tumor cells and interact with orally administered. Apoptotic LLC/OVA tumor cells were labeled withand then injected i.v. into mice. We followed the up-of CFSE-labeled apoptotic tumor cells in vivoph node or splenic DCs. As shown in Fig. 1B,

labeled apoptotic tumor cells were predominantlycytosed by splenic CD11c+ DCs. Consistent withus finding (33), only the CD8α+CD11c+ DC subsetspleen endocytosed the injected CFSE-labeled dyingcells. In experiments not depicted, we found thatFSE-labeled tumor cells were taken up by otherc− fractions of spleen, which were marked forb+ macrophages.etermine whether orally fed WGPs stimulate DC ac-n in vivo, groups of mice injected with fluoresceinKH26-labeled apoptotic LLC/OVA tumor cells i.v.treated with or without orally administered WGPs.ic DCs were analyzed by flow cytometry for the ex-on of a number of cell surface molecules. As indicat-Fig. 1C, injection of apoptotic tumor cells alone hadffect on the phenotype of the total CD11c positiveation with respect to the PBS control. In addition,pression levels of surface markers on total DCs werenally upregulated in total CD11c+ DCs in the pres-f WGP treatment (Fig. 1C, third row). Strikingly,GP treatment, surface markers such as costimula-

D80, CD86, and MHC class II molecules were signif-y upregulated in DCs that had captured apoptoticr cells, revealed by examining cells positive forc and PKH26 (Fig. 1C, compare fourth and fifthTherefore, the oral administration of WGPs allowshat capture tumor materials to activate in vivo.

treatment induces enhanced Ag-specific CD4 and-cell responsesdetermine if enhanced Ag-specific T-cell responsesbe induced by delivery of dying tumor cells ton WGP treatment, we first cocultured DCs in vitroapoptotic LLC/OVA tumor cells and OVA CD4 or
cells in the presence or absence of WGP treatment.treatment significantly augmented OVA Tg CD4
WGPsugge



proliferation in vitro (Fig. 2A). Similarly, CD8 T-celleration was also significantly increased (data notn). However, WGPs did not have direct effect onproliferation. To examine the possibility that WGPent could enhance the uptake of dying tumor cellss, DCs were cocultured with apoptotic tumor cells inresence or absence of WGPs. As shown in Fig. 2B,did not enhance DC-mediated phagocytosis of ap-

ic tumor cells in vitro.next examined whether orally administered WGPsce Ag-specific T-cell responses in vivo. Groups of miceinjected with apoptotic LLC/OVA tumor cells andlabeled OVA TCR Tg CD4 T cells (OT-II) were adop-transferred. As depicted in Fig. 3A, augmented CD4proliferation was induced in response to WGP treat-In addition, the number of activation/memory phe-es of CD4 T cells as defined as CD44highCD62Llowwasicantly increased in WGP-treated mice (Fig. 3B).rly, OVA CD8 T-cell proliferation was also augment-WGP treatment (Fig. 3C). Furthermore, the numberN-γ-producing CD8 T cells was significantly increasedGP-treated mice (Fig. 3D). These data stronglyst that particulate β-glucan WGP treatment leads toented Ag-specific CD4 and CD8 T-cell responses botho and in vivo.

treatment in vivo significantly delays tumoression with increased CD8-mediatedxic activity

r previous studies have showed that WGP in combi-with antitumor mAbs exhibited enhanced thera-

c efficacy (32, 36). To examine whether WGPent alone without exogenously administered anti‐r mAbs would have any therapeutic efficacy, we firstRAM-S-MUC1 lymphoma model. Groups of micegiven oral WGP treatment at different doses. WGPent with three different doses all caused delayed tu-rowth compared with saline treatment. Significancebserved when tumor-bearing mice received 200 andg WGP daily treatment. More importantly, WGPent with 400 μg daily dose also achieved significantnged survival (Fig. 4A).further confirm the therapeutic benefit provided byin other tumor models, groups of mice were im-d with LLC/OVA tumor cells. As shown in Fig. 4B,r-bearing mice treated with WGPs exhibited a signif-y smaller tumor burden compared with those treatedaline at days 23, 27, and 31 after tumor implanta-Further studies revealed that depletion of both CD4D8 T cells completely abrogated WGP-mediatedeutic efficacy (data not shown). Next, CD8 T-cellactivity was evaluated in tumor-bearing mice trea-

ith or without WGPs. As shown in Fig. 4C, miceut tumors had minimal killing activity. Mice with tu-but with PBS control treatment had ∼30% killing ac-Strikingly, tumor-bearing mice treated with oral
treatment had over 70% killing activity. These datast that WGP treatment can significantly delay tumor



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Oral WGPs migrate to the spleen and lymph node and interact with DCs. A, mice were fed orally with DTAF-WGPs (green). Spleen (Sp) andl lymph nodes (LN) were cryosectioned and stained with anti-CD11c mAb (red). B, mice were injected with 2 × 107 CFSE-labeled apoptoticA tumor cells or PBS. At 2 h after injection, spleen cells were harvested and stained with mAbs against CD11c and CD8α. Data show thattaking CFSE-positive apoptotic tumor cells are CD8α+. Cells were gated on CD11c+ population. C, groups of mice (n = 5) were treated withdaily for 7 d (400 μg/mouse). Fluorescein dye PKH26-labeled apoptotic LLC/OVA tumor cells (2 × 107 per mouse) were injected i.v. into C57Bl/6ive hours later, mice were sacrificed, and spleen cells were isolated and stained with mAbs against CD11c, CD40, CD80, CD86, MHC class II,evant isotype controls. Cells were gated on CD11c+ populations or CD11c+ and PKH26+ populations. Data show that costimulatory CD40,
CD86, and MHC class II molecules are significantly upregulated in DCs capturing tumor materials after WGP treatment. Numbers representluorescent intensity.
Clin Cancer Res; 16(21) November 1, 2010acrjournals.org 5157

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ssion, presumably via activating antitumor adaptiveresponses.

treatment in vivo significantly enhancesr-infiltrating IFN-γ–producing T cells and DCation within tumors and modulates theine secretion profilebecoming clear that tumors can actively subvert thene system through a variety of immune suppressivenisms within the tumor microenvironment (37, 38).termine whether WGP treatment had any effecte tumor microenvironment, we examined the typesiltrating cells and cytokine profiles within the LLC/umors. Tumors frommice treated with or without oral-inisteredWGPswere excised and live cells were stainedifferent mAbs including CD4, CD8, Gr-1 (granulo-, F4/80 (macrophages), CD11c (DC), and NK 1.1ral killer cells). The number of tumor-infiltrating
CD8, granulocytes, and natural killer cells was notcantly altered on WGP treatment (Fig. 5A; data not
mRNAmor m



). However, tumor-infiltratingCD4 andCD8T cells se-abundant IFN-γ on OVA stimulation in WGP-treatedcompared with those in PBS-treated mice (Fig. 5B).dition, the number of tumor-infiltrating DCs andphages was significantly increased on WGP treatmentA). More importantly, CD86 and MHC class II expres-evels were significantly increased on tumor-infiltratingfter WGP treatment in vivo (Fig. 5C). Similar resultsbserved in tumor-infiltrating macrophages (data not).t, we examined whether cytokine profiles in the tu-ilieu would be altered with or without WGP treat-

. To this end, LLC tumors from WGP-treated orated mice were excised and RNAs were extractedverse transcription-PCR. As shown in Fig. 5D, theof IL-4, IL-6, IL-10, IL-15, IL-17, transforming

h factor-β, tumor necrosis factor-α, and Foxp3 wereignificantly changed. Strikingly, IL-12 and IFN-γ

FigAg-in vDCphacocOVOVabsWGcell(50werB, CLLCwithabsCelcyt

s were both significantly increased within the tu-ilieu after WGP treatment. These data suggest that



WGPs significantly increasecific T-cell proliferationbut do not enhancediated apoptotic tumor cellytotosis. A, BMDCs werered with apoptotic LLC/mor cells and purified CD4T cells in the presence or

e of varying amounts ofDCs with CD4 Tg Tthe presence of OVA AgmL) with or without WGPssed as positive controls.E-labeled apoptoticA cells were coculturedDCs in the presence ore WGPs in vitro for 24 h.


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Enhanced Ag-specific T-cell responses on oral WGP in vivo treatment. A, groups of mice (n = 3) treated with or without WGPs (400 μg/daily/mouse

were injected i.v. with or without apoptotic LLC/OVA tumor cells. After 24 h, mice were adoptively transferred with CFSE-labeled OT-II CD4 T cells.ere continuously treated with or without WGPs for 5 d and sacrificed. Splenocytes were examined by flow cytometry. Cells were

n CFSE-positive cells. Percentage indicates proliferated cells. B, splenocytes were stained with mAbs against CD44 and CD62L. Cells

ted on CFSE-positive populations. C, groups of mice (n = 3) treated with or without WGPs were injected with or without apoptotic LLC/OVAells. After 24 h, mice were adoptively transferred with CFSE-labeled OT-I CD8 T cells. Mice were continuously treated with or without WGPs

and sacrificed. Splenocytes were examined by flow cytometry. Cells were gated on CFSE-positive cells. Percentage indicates proliferated cells. D,

ytes were restimulated with OVA (50 μg/mL) for 24 h and then stained for intracellular IFN-γ production. Cells were gated on CD8+ T cells.




Fig. 4.were imdaily) fos.c. witrecordeWGPssacrificcytotox

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WGP treatment significantly reduces tumor burden and prolongs survival with increased CD8 T-cell killing activity. A, groups of mice (n = 10)planted s.c. with RMA-S-MUC1 and, after 11 d, to allow tumor formation, were treated with oral WGPs at different doses (100, 200, and 400 μg,r 3 wk. Tumor-free survival was also monitored. Points, mean; bars, SEM. *, P < 0.05; **, P < 0.01. B, groups of mice (n = 12) were implantedh LLC/OVA tumor cells. After palpable tumors formed, mice were treated with or without orally administered WGPs for 3 wk. Tumor diameters wered at the indicated time. *, P < 0.05; **, P < 0.01. Points, mean; bars, SEM. B, tumor-bearing mice with LLC/OVA (n = 3) treated with or withoutfor 3 wk were adoptively transferred with OVA Class I peptide loaded CFSEhigh splenocytes with unloaded CFSElow splenocytes. Mice wereed after 24 h, and CFSE+ cells were gated and analyzed by flow cytometry. Data show that tumor-bearing mice treated with WGPs have the highest
icity against target cells compared with PBS-treated or naive mice.
ancer Res; 16(21) November 1, 2010 Clinical Cancer Research



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WGP treatment significantly increases IFN-γ-producing T cells and DC infiltration within the tumors and drives Th1 cytokine production in the tumorvironment. A, LLC/OVA tumor specimens (n = 5) from WGP-treated or untreated mice were prepared for single-cell suspensions. Tumor-infiltratingere assessed by flow cytometry. Data indicate that oral WGP treatment in vivo significantly increases DC and macrophage infiltration within the. Tumor samples were cryosectioned for immunohistochemistry staining with anti-CD11c mAb, which shows brown staining. Magnification, 200×.tions shown in this figure are representative tumor sections of 10 total tumor specimens. B, single cells from tumors treated with or without WGPsstimulated with OVA (50 μg/mL) for overnight and then performed surface staining with mAbs against CD4 or CD8 and intracellular IFN-γ staining.ere gated on CD4+ or CD8+ T-cell populations. C, single-cell suspensions were stained with mAbs against CD11c, CD40, CD80, CD86, and MHC

+
Cells were gated on a CD11c population. *, P < 0.05. D, RNAs from tumor specimens treated with or without WGPs (five tumors per group) wered and reverse-transcribed for reverse transcription-PCR for the indicated cytokines and transcriptional factor FoxP3. *, P < 0.05; **, P < 0.01.



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r a century ago, Sir William B. Coley observed spon-us tumor regression in some patients afflicted simul-usly with bacterial infection (39). Coley's toxins, are of heat-killed bacteria, were hypothesized to beve to induce tumor regression. In recent decades, itiscovered that bacteria contain conserved pathogen-ated molecular patterns that stimulate immune re-es (40). β-Glucans are major components of the cellf various fungi or bacteria and can be recognized asgen-associated molecular patterns by the mammali-ate immune system. Their mechanism of action intherapy occurs mainly via priming innate effectorphil CR3 to induce cytotoxicity of iC3b-opsonizedcells (41). Combined yeast-derived β-glucan with

mor mAb therapy is currently approved by the Foodrug Administration for its clinical investigation inI/II trials (42). However, recent studies have sug-that β-glucans also have a critical role in regulatingresponses (24, 30). Thus, the goal of the currentwas to investigate the possible interplays betweenan therapy and antitumor T-cell responses.iously, we showed that orally administered WGPsbe captured by gastrointestinal macrophages thatked into the BM, lymph node, and spleen (32).roperty was recently used by other investigators

ectively deliver proinflammatory cytokine silencingin mouse macrophages via engineering of glucan-sulated short interfering RNA particles (43). In this, we found that orally administered WGPs traf-into spleen and lymph nodes and directly inter-with DCs. More importantly, WGPs stimulatedc DCs that captured tumor materials to upregulatee accessory molecules, which are critical for T-celltion. Although it is surprising that WGPs only in-DC activation when these DCs have captured apo-tumor cells, it is possible that the DCs that

ct with WGPs are also the ones that preferentiallyre tumor cells. It is worth noting that DCs thated tumor material had upregulated surface acces-olecules even without WGP stimulation (data not). However, WGP treatment further activated thesehat had captured apoptotic tumor cells (Fig. 1C).less, WGP in vivo treatment subsequently leads to

ugmented Ag-specific CD4+ and CD8+ T-cell re-es. This is shown by two systems. Using thespecific T cell–adoptive transfer approach, wed that WGP treatment could stimulate Ag-specificand CD8 T-cell expansion and activation. In theVA tumor model, we further showed that aug-d antitumor CD4 and CD8 T-cell responses were
d in WGP-treated tumor-bearing mice, thus lead-the reduced tumor burden.
of ancomb



restingly, yeast zymosan β-glucan stimulates DCsrete abundant IL-10 but little IL-6 and IL-12, thusing immune tolerance (30, 44). In contrast, the cur-tudy shows that particulate yeast-derived β-glucanstimulate IL-12 and IFN-γ production within the

r microenvironment and significantly promote anti-r Th1 responses. These observed differences in DCne profiles and subsequent T-cell responses couldributed to the differential compositions of zymosanWGPs as we suggested previously (45). Zymosan

insoluble cell wall preparation and composes fewerans (12-14%). WGPs although purified from thestrain of yeast, S. cerevisiae, contain significantlyβ-glucan (>85%). We found that in vitro WGPsminantly trigger DCs to secrete IL-12 and tumorsis factor-α but low amounts of IL-6 and IL-10hat they significantly upregulate costimulatory mo-s and MHC class II (Supplementary Fig. S1). Theseations reaffirm the notion that different composi-of β-glucan preparations could lead to distinctical outcomes.becoming clear that the tumor microenvironmenta critical role in tumor immunotherapy, as tumorsctively subvert the immune system to establish anne-suppressive environment (37, 38). Therefore,ain challenge of tumor immunotherapy is to modu-e suppressive tumor microenvironment to favor thetion of strong antitumor immune responses. Wed that particulate β-glucan WGPs modulate the tu-icroenvironment toward Th1 responses via actively

ting DCs into the tumor milieu and further activat-em. The activated tumor-infiltrating DCs elicit aug-ed antitumor CD4 and CD8 T-cell responsesced by the fact that tumor-infiltrating T cells pro-abundant IFN-γ. However, it is unknown howtreatment engenders more DC infiltration within tu-It is possible that WGPs may mobilize DC precur-rom the BM. Indeed, previous studies have shown-glucan is capable of mobilizing hematopoieticprogenitor cells from the BM niche to the periphery7). It is also possible that DC trafficking into tumorsulated by different subset of T cells. A recent studyd that Th17 cells promote DC infiltration into lungr tissues (27).r previous study showed that orally administeredβ-glucan synergizes with antitumor mAbs to induceicant tumor regression and achieve long-term tumor-rvival (32). These effects were ascribed to the in vivoriming of effector neutrophils, because their thera-efficacy was significantly decreased in CR3-deficientHowever, WGP treatment only showed significanterm tumor-free survival with respect to untreatedb-treated animals in RAM-S-MUC1 tumor model,is efficacy is independent of CR3 (32), which is con-t with our current findings. Based on the current re-we speculate that this effect is due to the activation
titumor T-cell responses elicited by WGPs. Thus,ined therapy with β-glucan WGPs not only elicits



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/National Cancer Institute grants RO1 CA86412 and RO1CA150947tucky Lung Cancer Research Program.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 03/30/2010; revised 06/22/2010; accepted 06/30/2010;ed OnlineFirst 09/20/2010.
n: scientific consultant, Biothera.
rencesler IC, Mankowski ZT, Fisher ME. The effect of yeast polysacchar-s on mouse tumors. Cancer Res 1963;23:201–8.Luzio NR, Williams DL, McNamee RB, Edwards BF, Kitahama A.mparative tumor-inhibitory and anti-bacterial activity of solubleparticulate glucan. Int J Cancer 1979;24:773–9.

nter JT, Meltzer MS, Ribi E, et al. Glucan: attempts to demonstraterapeutic activity against five syngeneic tumors in guinea pigs ande. J Natl Cancer Inst 1978;60:419–24.igne LM, O'Brien XM, Kim M, Janowski JW, Albina JE, Reichner. Integrin engagement mediates the human polymorphonuclearkocyte response to a fungal pathogen-associated molecularttern. J Immunol 2007;178:7276–82.sley HE, Tobias B, Pretus HA, et al. NMR spectral analysis of ater-insoluble (1->3)-β-D-glucan isolated from Saccharomycesevisiae. Carbohydr Res 1994;258:307–11.ss GD. Regulation of the adhesion versus cytotoxic functions ofMac-1/CR3/αMβ2-integrin glycoprotein. Crit Rev Immunol 2000;197–222.merman JW, Lindermuth J, Fish PA, Palace GP, Stevenson TT,Mong DE. A novel carbohydrate-glycosphingolipid interaction be-en a β-(1–3)-glucan immunomodulator, PGG-glucan, and lacto-ceramide of human leukocytes. J Biol Chem 1998;273:22014–20.e PJ, Kelley JL, Kogan G, et al. Human monocyte scavengereptors are pattern recognition receptors for (1->3)-β-D-glucans.eukoc Biol 2002;72:140–6.own GD, Gordon S. Immune recognition. A new receptor forlucans. Nature 2001;413:36–7.own GD, Herre J, Williams DL, Willment JA, Marshall AS,rdon S. Dectin-1 mediates the biological effects of β-glucans.xp Med 2003;197:1119–24.ntner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM.llaborative induction of inflammatory responses by dectin-1 andll-like receptor 2. J Exp Med 2003;197:1107–17.ornton BP, Vetvicka V, Pitman M, Goldman RC, Ross GD. Analysisthe sugar specificity and molecular location of the β-glucan-ding lectin site of complement receptor type 3 (CD11b/CD18).munol 1996;156:1235–46.Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, Ross GD. Thelucan-binding lectin site of mouse CR3 (CD11b/CD18) and itsction in generating a primed state of the receptor that mediatesotoxic activation in response to iC3b-opsonized target cells.munol 1999;162:2281–90.

n J, Vetvicka V, Xia Y, et al. β-Glucan, a “specific” biologic re-onse modifier that uses antibodies to target tumors for cytotoxicognition by leukocyte complement receptor type 3 (CD11b/18). J Immunol 1999;163:3045–52.ng F, Hansen RD, Yan J, et al. β-Glucan functions as an adjuvantmonoclonal antibody immunotherapy by recruiting tumoricidalnulocytes as killer cells. Cancer Res 2003;63:9023–31.ndorf DJ, Yan J, Ross GD, et al. C5a-mediated leukotriene B4-plified neutrophil chemotaxis is essential in tumor immunotherapyilitated by anti-tumor monoclonal antibody and {β}-glucan. J Im-

yk-phosphatidylinositol 3-kinase pathway. J Immunol 2006;177:61–9.dak S, Koehne G, Vickers A, O'Reilly RJ, Cheung NK. Rituxi-b therapy of lymphoma is enhanced by orally administered>3),(1->4)-D-β-glucan. Leuk Res 2005;29:679–83.lvador C, Li B, Hansen R, Cramer DE, Kong M, Yan J. Yeast-rived {β}-glucan augments the therapeutic efficacy mediated byti-vascular endothelial growth factormonoclonal antibody in humanrcinoma xenograft models. Clin Cancer Res 2008;14:1239–47.ong W, Hansen R, Li B, et al. Effect of yeast-derived β-glucan innjunction with bevacizumab for the treatment of human lung ade-carcinoma in subcutaneous and orthotopic xenograft models.mmunother 2009;32:703–12.B, Allendorf DJ, Hansen R, et al. Combined yeast {β}-glucan andtitumor monoclonal antibody therapy requires C5a-mediated neu-phil chemotaxis via regulation of decay-accelerating factor CD55.ncer Res 2007;67:7421–30.eung NK, Modak S. Oral (1->3),(1->4)-β-D-glucan synergizes withtiganglioside GD2 monoclonal antibody 3F8 in the therapy of neu-lastoma. Clin Cancer Res 2002;8:1217–23.eung NK, Modak S, Vickers A, Knuckles B. Orally administeredlucans enhance anti-tumor effects of monoclonal antibodies.ncer Immunol Immunother 2002;51:557–64.ibundGut-Landmann S, Gross O, Robinson MJ, et al. Syk- andRD9-dependent coupling of innate immunity to the induction ofelper cells that produce interleukin 17. Nat Immunol 2007;8:0–8.ibundgut-Landmann S, Osorio F, Brown GD, Reis e Sousa C.mulation of dendritic cells via the dectin-1/Syk pathway allowsming of cytotoxic T-cell responses. Blood 2008;112:4971–80.orio F, LeibundGut-Landmann S, Lochner M, et al. DC activateddectin-1 convert Treg into IL-17 producers. Eur J Immunol 2008;:3274–81.rtin-Orozco N, Muranski P, Chung Y, et al. T helper 17 cellsmote cytotoxic T cell activation in tumor immunity. Immunity09;31:787–98.yczek I, Banerjee M, Cheng P, et al. Phenotype, distribution,neration, and functional and clinical relevance of Th17 cells inhuman tumor environments. Blood 2009;114:1141–9.

yczek I, Wei S, Szeliga W, Vatan L, Zou W. Endogenous IL-17ntributes to reduced tumor growth and metastasis. Blood 2009;4:357–9.lon S, Agrawal S, Banerjee K, et al. Yeast zymosan, a stimulus forR2 and dectin-1, induces regulatory antigen-presenting cells andmunological tolerance. J Clin Invest 2006;116:916–28.ini R, Torosantucci A, Romagnoli G, et al. {β}-Glucan of Candidaicans cell wall causes the subversion of human monocyte differ-tiation into dendritic cells. J Leukoc Biol 2007.ng F, Yan J, Baran JT, et al. Mechanism by which orally adminis-ed β-1,3-glucans enhance the tumoricidal activity of antitumornoclonal antibodies in murine tumor models. J Immunol 2004;3:797–806.
K, Idoyaga J, Charalambous A, et al. Innate NKT lymphocytes
nfer superior adaptive immunity via tumor-capturing dendriticlls. J Exp Med 2005;202:1507–16.

Clin Cancer Res; 16(21) November 1, 2010 5163



34. Dincela c

35. Dinauganc

36. Yaβ-gtre

37. CroSccelmic

38. Rastr25:

39. Hotum200

40. Herec

41. Liu

witMo

42. TaesanJ C

43. Aoing20

44. KamuJ I

45. Limuvia20

46. Crproma

Li et al.

Clin C5164

Do


g C, Cai Y, Marroquin J, Ildstad ST, Yan J. Plasmacytoid dendriticls regulate autoreactive B cell activation via soluble factors and inell-to-cell contact manner. J Immunol 2009;183:7140–9.g C, Wang L, Marroquin J, Yan J. Targeting of antigens to B cellsments antigen-specific T-cell responses and breaks immune toler-e to tumor-associated antigen MUC1. Blood 2008;112:2817–25.n J, Allendorf DJ, Brandley B. Yeast whole glucan particle (WGP)lucan in conjunction with antitumour monoclonal antibodies toat cancer. Expert Opin Biol Ther 2005;5:691–702.ci DO, Zacarias Fluck MF, Rico MJ, Matar P, Rabinovich GA,harovsky OG. Dynamic cross-talk between tumor and immunels in orchestrating the immunosuppressive network at the tumorroenvironment. Cancer Immunol Immunother 2007;56:1687–700.binovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressiveategies that are mediated by tumor cells. Annu Rev Immunol 2007;267–96.ption Cann SA, van Netten JP, van Netten C. Dr William Coley andour regression: a place in history or in the future. Postgrad Med J3;79:672–80.
ine H, Ulmer AJ. Recognition of bacterial products by toll-likeeptors. Chem Immunol Allergy 2005;86:99–119.J, Gunn L, Hansen R, Yan J. Combined yeast-derived β-glucan
47. Crcoinj



h anti-tumor monoclonal antibody for cancer immunotherapy. Expl Pathol 2009;86:208–14.mayo ME, Bautista JB, Flores ML, et al. A phase Ib/2, dose-calating, safety, and efficacy study of imprime PGG, cetuximabd irinotecan in patients with advanced colorectal cancer (CRC).lin Oncol 2009;27:Abstract: e15062.uadi M, Tesz GJ, Nicoloro SM, et al. Orally delivered siRNA target-macrophage Map4k4 suppresses systemic inflammation. Nature

09;458:1180–4.rumuthil-Melethil S, Perez N, Li R, Vasu C. Induction of innate im-ne response through TLR2 and dectin 1 prevents type 1 diabetes.mmunol 2008;181:8323–34.B, Cramer D, Wagner S, et al. Yeast glucan particles activaterine resident macrophages to secrete proinflammatory cytokinesMyD88- and Syk kinase-dependent pathways. Clin Immunol

07;124:170–81.amer DE, Wagner S, Li B, et al. Mobilization of hematopoieticgenitor cells by yeast-derived β-glucan requires activation oftrix metalloproteinase-9. Stem Cells 2008;26:1231–40.amer DE, Allendorf DJ, Baran JT, et al. {β}-Glucan enhancesmplement-mediated hematopoietic recovery after bone marrow
ury. Blood 2006;107:835–40.



2010;16:5153-5164. Published OnlineFirst September 20, 2010.Clin Cancer Res Bing Li, Yihua Cai, Chunjian Qi, et al. T-Cell Responses in CancerTumor-Capturing Dendritic Cells and Improves Antitumor

-Glucan ModulatesβOrally Administered Particulate

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Cancer Therapy: Preclinical Clinical Cancer Research Orally ... · previously (34). Cultured BMDCs at day 7 were used for experiments. BMDCs were further purified with anti-CD11c