Stromal Cell PD-L1 Inhibits CD8þ T-cell AntitumorImmune Responses and Promotes Colon CancerGrace O'Malley1,2, Oliver Treacy1,2, Kevin Lynch2, Serika D. Naicker2,Niamh A. Leonard1,2, Paul Lohan2, Philip D. Dunne3, Thomas Ritter2,Laurence J. Egan1, and Aideen E. Ryan1,2
Abstract
Stromal cells of mesenchymal origin reside below theepithelial compartment and provide structural support in theintestine. These intestinal stromal cells interact with both theepithelial cell compartments, as well as infiltrating hemato-poietic immune cells. The importance of these cells in regu-lating immune homeostasis during inflammation is wellrecognized. However, little is known about their function andphenotype in the inflammatory tumor microenvironment.Using a syngeneic, immunogenic model of colorectal cancer,we showed that TNFa-initiated inflammatory signaling inCT26 colorectal cancer cells selectively induced PD-L1 expres-sion in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1potentiated enhanced immunosuppression, characterized byinhibition of activated CD8þ granzyme B-secreting T cells
in vitro, and the inhibition of CD8þ effector cells wasassociated with enhanced tumor progression. Stromal cellimmunosuppressive and tumor-promoting effects couldbe reversed with administration of anti–PD-1 in vivo. Wevalidated our findings of stromal cell CD274 expression in twocohorts of clinical samples and also observed PD-L1 inductionon human stromal cells in response to exposure to the inflam-matory secretome fromhuman colon cancer cells, irrespective ofmicrosatellite instability. Collectively, our data showed thattumor-associated stromal cells support T-cell suppression byPD-L1 induction, which is dependent on colon cancer inflam-matory signaling. Our findings reveal a key role ofmesenchymalstromal cells PD-L1 in suppression of CD8þ antitumor immuneresponses and potentiation of colorectal cancer progression.Cancer Immunol Res; 6(11); 1–16. �2018 AACR.
IntroductionColon cancer development and metastasis is a multistep pro-
cess, during which accumulation of genetic mutations drivestumor development through acquisition of hallmarks, includingthe ability to evade immune surveillance (1). Colon tumors arecomposed of numerous cellular components that can facilitate animmunosuppressive microenvironment, including immune,stromal, and endothelial cells (2–5). In colon cancer, the type,density, and location of immune cells within tumors can predictclinical outcome (6), and colon cancer patients without tumorrecurrence are shown to have higher immune cell densities (CD3,
CD8, granzyme B) than patients whose tumors recurred aftertreatment (6). These observations highlight the need to betterunderstand the cellular andmolecularmechanisms that dictate animmunosuppressive microenvironment in colon cancer.
In the colon cancer microenvironment, activated T cells trafficfrom the blood or lymph nodes into the tumor. The stromal cellsin the colon are a heterogeneous population (7, 8), with a largeproportion being of mesenchymal origin. They are positionedbetween the epithelial cells and the underlying vasculature andcan passively or actively impair immune cell trafficking andactivation. Stromal cell influence in the tumormicroenvironmenthas been highlighted in several studies that identify a stromal cellsignature from colon tumors that is associated with tumor pro-gression and a poorer prognosis in colon cancer patients (9).However, it remains poorly understood if the prognosis associ-atedwith these signatures is due to the inherent tumor-promotingor immunomodulatory potential of mesenchymal cells, whichconstitute a significant proportion of stromal cells in the intestinalmicroenvironment. These findings highlight the need to betterunderstand the immunomodulatory role of the tumor stromalcompartment to improve the development of effective stroma-targeting strategies.
Inflammation can promote tumorigenesis and is recognized asone of the hallmarks of cancer (1, 10). Inflammatory boweldisease is causally associated with colon cancer promotion(11). The transcription factor NF-kB, a key regulator of bothinflammation and cancer, regulates the expression ofmany proin-flammatory chemokines and cytokines and is associatedwith cancer progression and metastasis (8, 12). Many NF-kB–dependent soluble factors, including TNFa, can directly activate
1Discipline of Pharmacology and Therapeutics, School of Medicine, College ofMedicine, Nursing and Health Sciences, National University of Ireland Galway,Galway, Republic of Ireland. 2Regenerative Medicine Institute (REMEDI), Schoolof Medicine, College of Medicine, Nursing and Health Sciences, National Uni-versity of Ireland Galway, Galway, Republic of Ireland. 3Centre for CancerResearch and Cell Biology, Queens University Belfast, Belfast, United Kingdom.
Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).
O. Treacy, K. Lynch, and S.D. Naicker contributed equally to this article.
L.J. Egan and A.E. Ryan share senior authorship of this article.
CorrespondingAuthor:AideenE. Ryan, BMS-1023, First Floor South, BiomedicalSciences Building, North Campus, Dangan, NUI Galway, National University ofIreland Galway, Galway, Ireland. Phone: 091 495107; E-mail:[email protected]
stromal cells to inhibit adaptive and innate immune effectormechanisms (13). Mesenchymal stromal cells (MSC) can sup-press effector T lymphocytes, but little is known about how thesestromal cell properties are altered in the inflammatory colontumor microenvironment (14).
One important mechanism by which tumors avoid clear-ance by the immune system is by upregulating the expressionof immunomodulatory ligands. In colorectal cancer, pro-grammed death-ligand 1 (PD-L1) expression on tumor cellsand tumor-infiltrating lymphocytes is of particular importanceto this immune escape phenotype (15, 16). The receptor forPD-L1, PD-1, is expressed on activated T cells and, whenbound by the ligand, results in downregulated proliferationand inhibition of effector function (15). The PD-1/PD-L1pathway represents an adaptive immune resistance mecha-nism that is exerted by tumor cells in response to endogenousantitumor activity. An antibody targeting PD-1 has beengranted FDA approval for metastatic colon cancer (17).Although the PD-1/PD-L1 signaling axis is important in colo-rectal cancer, anti–PD-1 therapy is not always effective in thissetting, with some patients experiencing disease progressionfollowing treatment (18). Efforts to better stratify patients foranti–PD-1 immunotherapy have relied on the measurement oftumor or immune cell PD-L1 expression, but reports show thatsome patients deemed tumor PD-L1–negative respond posi-tively to anti–PD-1 therapy (19).
The stromal compartment of the tumormicroenvironment hasreceived little attention to date in terms of stratifying patients forimmunotherapy despite the known immunomodulatory poten-tial of these cells. We, therefore, hypothesized that stromal cells inthe inflammatory colon tumor microenvironment can directlymodulate T-cell–mediated antitumor immunity through cellcontact–dependent mechanisms. We showed that stromal cellPD-L1 expression induced by TNFa signaling in colon cancer cellsled to decreased CD8þ T-cell proliferation, activation, and pro-motion of colon cancer growth in vivo.
Materials and MethodsAnimals
Eight- to 14-week-old female Balb/c mice were purchased fromCharles River. Experimental animals were housed in a specificpathogen-free facility and fed a standard chowdiet. All proceduresperformed were conducted in a fully accredited animal housingfacility at NUI Galway under a license granted by the HealthProducts Regulatory Authority, Ireland, andwere approved by theNUI Galway Animal Care Research Ethics Committee.
Cell culture, MSC isolation, and conditioningMouse colon adenocarcinoma cells CT26, derived from
Balb/c mice, were purchased from the European Collection ofCell Cultures (ECACC) and cultured in DMEM (Biosciences-Gibco) supplemented with 10% fetal bovine serum (FBS;Sigma) and 1% penicillin/streptomycin (Sigma). To assess theeffects of CT26 NF–kB signaling, stable CT26 clones expressinga mutant form of the human I8B-a with serine 32 and 36mutated into alanine (I8B-a SR) were generated as previouslydescribed (4). Human colon cancer cell lines HCT116 andHT29 were purchased from the ATCC and cultured in McCoys5A medium (Sigma) supplemented with 10% FBS (Fisher,Hyclone), 1% L-glutamine, and 1% penicillin/streptomycin
(Sigma). Mouse and human cell master stocks were authenti-cated by ECACC/ATCC, confirmed mycoplasma negative(MycoAlert, Lonza), expanded, frozen, and used within 15passages of testing for all subsequent experiments.
For murine MSC isolation, Balb/c mice were euthanized byCO2 inhalation, and the femur and tibia were removed, cleaned,and placed inMSC culture medium; MEM-a (Biosciences-Gibco)supplemented with 10% FBS (Fisher, Hyclone), 10% Equineserum (Fisher, Hyclone), and 1% penicillin/streptomycin(Sigma). MSC were flushed from the bones, filtered, and platedat a density of 9 � 105 per cm2 in a T175 flask. Cells wereincubated at 37�C, 5%CO2, and nonadherent cells were removed24hours later. This processwas repeated3 times perweekuntil thecells reached confluency. MSC were characterized according tothe criteria published by the ISCT cell-surface characterization(Supplementary Fig. S1A), and trilineage differentiation wascarried out at passage 4 (Supplementary Fig. S1B–S1D). Allsubsequent experiments were carried out with mouse MSCbetween passages 5 and 14.
HumanMSC (hMSC) were isolated from the bone marrow ofthree healthy volunteers at Galway University Hospital underan ethically approved protocol (NUIG Research Ethics Com-mittee, Ref: 08/May/14) according to a standardized procedure.Written consent was obtained from the volunteers. Briefly,bone marrow cell suspensions were layered onto a Ficolldensity gradient, and the nucleated cell fraction was collected,washed, and resuspended in MSC culture medium. After 24hours of cultivation, nonadherent cells were removed, freshmedium was added, and individual colonies of fibroblast-likecells were allowed to expand and approach confluence prior topassage. hMSC were grown in alpha MEM (Gibco, Invitrogen)supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin–streptomycin, and fibroblast growth factor 2 (FGF2, 1 ng/mL;Sigma-Aldrich).
For MSC tumor conditioning (MSCTCM), CT26, HCT116,and/or HT29 cells were seeded in a T175 flask (Nunc, Fisher) ata density of 1 � 106 cells per flask in 25 mL of the correspondingtumor cell culture medium. Cells were left to grow at 37�C at 5%CO2 (normoxia) for a total of 72 hours, at which point condi-tioned medium was collected, spun at 1,000 � g to pellet anycellular debris, and stored at �80�C. For TNFa conditioning(MSCTNF-TCM), TNFa (100 ng/mL; PeproTech) was added totumor cell cultures 24 hours prior to collection of tumor cellmedium. For conditioning mouse MSC, cells were seeded at adensity of 0.03� 106 cells per well of a 6-well plate in 2 mL MSCculture medium. Human MSC were seeded at a density of 0.1 �106 cells per well of a 6-well plate. Twenty-four hours afterseeding, the culture medium was removed and replaced with40% fresh MSC medium and 60% tumor-conditioned medium.Fresh DMEM (MSCControl) and DMEM with TNFa (100 ng/mL;MSCTNF) were included as controls. MSC were analyzed at 24and 72 hours after addition of conditioned medium. In vitroimmunosuppression assays and in vivo experiments, describedbelow, were all carried out using MSC that had been conditionedfor 72 hours.
NF-kB-Luc; pNF-kB-Luc plasmid; Stratagene) were used in thisstudy. CT26/EV or CT26/I8B-a SR cells were generated andvalidated as previously described (4), plated in a 96-well plate,
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Cancer Immunol Res; 6(11) November 2018 Cancer Immunology ResearchOF2
and cotransfected with 500 ng of pNF-kB-Luc or 500 ng of pLuccontrol vector (Stratagene) and 50 ng of RSV-pRL reporter(Promega Corp.). Twenty-four hours after transfection, cells weretreated with TNFa at doses of 10 to 100 ng/mL for 24 hours.NF-kB activity was determined by analysis of luciferase activitywith theDual-Glo Luciferase Reporter Assay System (Promega) tovalidate reduced NF-kB activity in CT26/I8B-a SR cells (Supple-mentary Fig. S2A).
in pGFP-C-shLenti plasmids (CD274 shRNA Lenti-plasmid) andone scrambled negative control noneffective shRNA cassette inpGFP-C-shLenti plasmid (scramble shRNA Lenti-plasmid) werepurchased from OriGene (OriGene Technologies; Cat. No.TL503436). Lentiviruses were generated by cotransfection of293 T cells with either CD274 shRNA or scramble shRNALenti-plasmids, as well as with packaging (psPAX2.2), envelope(pMD2.G), and additional (pRSV-rev) plasmids using a standardprotocol (Invitrogen). Supernatants were harvested 48 and 72hours after transfection, combined, filtered through 0.45-mmporesize filters, and frozen at �80�C until required.
Lentiviral transduction of MSCBalb/c MSC were removed from liquid nitrogen storage and
passaged twice before transduction. Because pGFP-C-shLentiplasmids encode a puromycin resistance gene, MSC were treatedwith 7 mg/mL of puromycin (Gibco-Biosciences) for 96 hours,followed by 72 hours recovery in fresh MSC media. Followingthis, cells were imagedbyfluorescentmicroscopy to identifyGFPþ
cells (Supplementary Fig. S4A). GFP expression was observed inboth the CD274 shRNA– and scramble shRNA–transduced MSC,demonstrating successful transduction (Supplementary Fig. S4A).
RNA isolation and qRT-PCRTo determine if successful knockdown of PD-L1 was
achieved, CD274 shRNA– and scramble shRNA–transducedMSC were centrifuged at 800 � g, supernatants were removed,and RNA was isolated from cell pellets using the Isolate II RNAMiniKit (Bioline) according to the manufacturer's instructions.RNA was resuspended in 40 mL of nuclease-free water andquantified by NanoDrop. cDNA was synthesized using Rever-tAid H Minus Reverse Transcriptase (Fermentas) with randomprimers. Two-step qRT-PCR was performed to determine themRNA expression of CD274 (IDT Primetime qPCR assay:Mm.PT.58.11921659, Integrated DNA Technologies) by com-paring Ct values with that of the housekeeping gene GAPDH(Primetime qPCR assay: Mm.PT.39a.1, Integrated DNA Tech-nologies). Quantitative real-time PCR was performed accordingto the standard program on the ABI Step-one machine (AppliedBiosystems). CD274 mRNA expression in CD274 shRNA– andscramble shRNA–transduced MSC are illustrated in Supple-mentary Fig. S4B.
Validation of tumor-conditioned shRNA PD-L1 MSCMSC were seeded at a concentration of 1� 105 cells per 6-well
in 2 mL of mouse MSC media and allowed to adhere overnight.Lentivirus-containing supernatants from the different prepara-tions weremixed withmouseMSCmedia at a ratio of 1:5 (mouseMSC media:lentiviral supernatant) to a final volume of 2 mL perwell. Plateswere incubated at 37�C, 5%CO2 for 24hours. After 24
hours, the virus-containing media were removed and replacedwith fresh mouse MSC media mixed with conditioned mediumfrom CT26 cells preactivated with TNFa (MSCTNF-TCM; 40%MSCmedia and 60% TNFa TCM) and incubated for 24 hours. To testthe transduction efficiency, 48 hours after transductionGFPþ cellswere analyzed by flow cytometry (Supplementary Fig. S4D). Inthe same experiment, successful inhibition of PD-L1 expressionon PD-L1 shRNA MSC was confirmed (Supplementary Fig. S4Eand see Supplementary Fig. S4C for gating strategy).
and cervical lymph nodes, as well as spleens, were harvested fromhealthy animals following CO2 asphyxiation. Single-cell suspen-sions were obtained by mechanical disruption (using a 1-mLsyringe plunger) of the tissue in culture medium: RPMI 1640(Fisher, Lonza) supplementedwith 10%FBS (Sigma), 1%sodiumpyruvate, 1% nonessential amino acids, L-glutamine, 1% peni-cillin/streptomycin (all Sigma), and 0.01% b-mercaptoethanol.Cells were washed, resuspended in PBS, and erythrocytes lysedusing ammonium–chloride–potassium lysing buffer (Sigma-Aldrich) for 5 minutes on ice. Cells were resuspended in culturemedium, and a combinationof 90% lymphocyte/10% splenocytewas used for immunosuppression assays.
To assess proliferation, cells were stained with carboxyfluor-escein diacetate succinimidyl ester (CFSE) using the CellTracecell proliferation kit (Invitrogen). For assessment of effects ofshRNA MSC, lymphocytes were stained with cell trace violet(CTV) using a CellTrace Violet Cell Proliferation Kit (ThermoFisher Scientific) in order to exclude MSC GFP expression fromanalysis. Cells were counted and resuspended in 1 mL PBS at aconcentration of 1 � 107 cells/mL. CFSE and CTV were recon-stituted in dimethyl sulfoxide, and 2 mL of reconstituted dyewas added for each 1 mL of BSA/PBS. Cells were protected fromlight and incubated at 37�C for 6 minutes. Ice-cold culturemedium was added to neutralize the CFSE, and cells werewashed twice more. Cells were then activated using CD3/CD28Mouse T-Activator Dynabeads (Life Technologies) for the dura-tion of the 72-hour coculture.
CFSE-labeled and activated cells (0.1 � 106) from whole-cellisolates (90% lymphocyte/10% splenocyte) were coculturedwith na€�ve or tumor-conditioned Balb/c MSC or TNFa tumor-conditioned PD-L1 shRNA– and scramble shRNA–transducedMSC at a ratio of 1MSC:50 or 10 lymphocytes in a 96-well roundbottom cell culture plate (Sarstedt). After 72 hours, medium wasremoved from the cocultures, aliquoted, and stored at�80�C forfurther analysis by ELISA. Cells were resuspended in FACS buffer(PBS supplemented with 2% FBS and 0.05% sodium azide) andwere incubated with CD8a (APC, Clone 53-6.7; BioLegend) orCD4 (PerCP-Clone GK1.5; BioLegend) and/or CD25 (PE-clonePC61; BioLegend) for 15 minutes at 4�C. Cell viability wasassessed following resuspension in 100 mL of FACS buffer byaddition of the viability dye SYTOXblue (Thermo Fisher, S34857;Supplementary Fig. S2B) according to the product instructions.Cells were washed twice, and proliferation was measured using aFACSCanto cytometer (Becton Dickinson). Data were analyzedusing FlowJo software (Version X, TreeStar Inc.). T-cell suppres-sion was calculated according to the formula: 100� ((% positivecontrol divided/% sample divided) � 100) for all populationdoublings greater than the second generation (SupplementaryFig. S2C and S2D).
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IFNg, TNFa, and granzyme B enzyme-linked immunosorbentassays (ELISA)
Supernatants from MSC and T-cell cocultures were analyzedusing Ready-SET-Go! ELISA kits (Affymetrix, eBioscience) forsecretion of IFNg , TNFa, and granzyme B. Flat-bottom 96-wellELISA plates were coated overnight at 4�C with capture antibodyaccording to themanufacturer's protocol. The followingmorning,plates were washed with wash buffer (1� PBS with 0.05%Tween-20), and plates were blocked using the supplied assaydiluent for 1 hour and then washed. Standards, as supplied withthe assays and 100 mL of samples (1:2 dilution), were added to theplates and incubated for 24 hours at 4�C. Following 3 washes,detection antibody (100 mL) was added and incubated at roomtemperature for 1 hour. Following washing, 100 mL avidin horse-radish peroxidase (HRP) was added for 30minutes and washed afurther 3 times. Tetramethylbenzidine substrate solution (100mL)was then added and left at room temperature for 15 minutesbefore 50 mL stop solution (2N sulfuric acid) was added, and theplates were read at 450 and 570 nmon aWallac 1420 plate reader(PerkinElmer).
Subcutaneous tumor modelTumors were induced in 8- to 14-week-old female Balb/c
mice by subcutaneous injection into the left flank of 2 � 105
CT26 cells � 0.5 � 105 MSC (�/þ in vitro tumor precondi-tioning MSCControl/MSCTNF-TCM) in a total volume of 100 mLPBS. Animals receiving PD-1 antibody therapy received anintraperitoneal injection of 200 mg of anti–PD-1 (cloneRMP1-14; Bio X Cell, 2B Scientific) in 100 mL PBS on days 7and 14 after tumor induction. Tumor growth was monitoreddaily until sacrifice on day 21. At day 21, animals were eutha-nized, and tumors were harvested from left flank and, wheretumor invasion had occurred as determined by visual tumordeposits, the peritoneal cavity. Tumor measurements weretaken using digital calipers, and tumor volume was calculatedaccording to the rational ellipse formula: (M1^2 � M2 � p/6).Images of tumors were taken of tumor tissue dissected fromanimals at day 21.
Cell-surface characterization of MSC or CT26 cells by flowcytometry
MSC or CT26 cells were trypsinized, counted, and washed inFACS buffer (PBS supplemented with 2% FBS and 0.05% sodiumazide) prior to staining. Staining was carried out by incubatingthe cells with the antibody of interest in FACS buffer at 4�C for 10minutes. Following this incubation, cells were washed twice inFACS buffer, stained with the viability dye SYTOX blue (ThermoFisher, S34857), and analyzed straight after or prepared forintracellular staining (protocol below). Antibodies used wereas follows: MHC-I (APC, clone SF1-1.1), MHC-II (FITC, clone39-10-8), CD80 (PE, clone 16-10A1), CD86 (PE, clone PO3),PD-L1 (APC, clone 10F.9G2), PD-L2 (PE, clone TY25), and FasL(biotin with streptavidin–PE). All antibodies were purchasedfrom BioLegend. Streptavidin was purchased from eBioscience.For characterization, 100,000 CT26 or MSC were stained permarker.
Analysis of tumors by flow cytometryTo analyze tumor immune cell infiltrates, tumors were
digested in 1 mL HBSS (Gibco-Biosciences) containing150 U/mL collagenase IV (Biosciences) and 200 U/mL DNase
(Sigma). Samples were left at 37�C for 2 hours and then filteredthrough 40-mm cell strainers and washed with PBS. Single-cellsuspensions were counted and stained with markers of interest:CD3 (FITC, clone 17A2), CD8 (APC, clone 53-6.7), CD25 (PE,clone PC61), CD4 (APC, clone GK1.5), all BioLegend. Cell-surface staining was carried out as before using 1 � 106 cells persample and appropriate FMOs were used to ensure stainingaccuracy.
To stain for intracellular granzyme B (PE, clone REA226;Miltenyi), cells were washed twice after surface staining had beencompleted and fixed [2% paraformaldehyde (Sigma) for 10minutes at 4�C] for intracellular staining. Cells were then incu-bated with granzyme B antibody diluted in permeabilizationbuffer (1% BSA/PBS with 0.5% saponin) according to the man-ufacturer's instructions. Samples were again incubated at 4�C for10 minutes, washed twice in FACS buffer, and analyzed on aFACSCanto II.
Transcriptional data setsGene-expression profiles from two independent colorectal
cancer data sets were downloaded from the NCBI Gene-Expres-sion Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under acces-sion numbers GSE35602 and GSE70468. GSE35602 containsmicroarray profiles separately profiled from laser-capture micro-dissected stroma or epithelium regions from 13 colorectal cancerprimary tumors (20). GSE70468 contains microarray profilesfrom primary fibroblasts derived using colon primary tumor andmorphologically normal colonic mucosa tissue isolated fromfresh colorectal cancer resection material. Both studies indicatethat they were performed after approval by an institutional reviewboard (IRB), and informedwritten consentwas obtained from thesubjects (20, 21).
Bioinformatics analysisPartek Genomics Suite software (version 6.6; Partek Inc.) was
used for analysis of the independent data sets. For the purpose ofclustering, datamatrices were standardized to themedian value ofprobe set expression. Standardization of the data allows forcomparison of expression for different probe sets. Followingstandardization, two-dimensional hierarchical clusteringwas per-formed (samples � probe sets/genes). Euclidean distance wasused to calculate the distance matrix, a multidimensional matrixrepresenting the distance from each data point (probe set-samplepair) to all the other data points. Ward's linkage method wassubsequently applied to join samples and genes together, with theminimum variance, to find compact clusters based on the calcu-lated distance matrix.
Statistical analysisStatistical analysis was carried out using GraphPad Version X
(GraphPad Software). Data were assessed for normal distribu-tion using D'Agostino–Pearson omnibus normality test. Datasets with two groups were analyzed using an unpaired t test.Data sets with more than two groups were analyzed by ordinaryone-way ANOVA followed by the Tukey multiple comparisonstest. For analysis of correlation, Pearson correlation coefficientwas calculated. To test for a trend in tumor invasion data, a c2
test was carried out, and to test for the effect of antibodytreatment on individual treatment groups, Fisher exact testwas used. Results were considered statistically significant atP < 0.05.
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ResultsInflammatory tumor-conditioned stromal cells inhibit T-cellproliferation and activation
A large proportion of the stromal component of the healthycolon and colon tumor microenvironment are of mesenchymalorigin, which is an important barrier to overcome for infiltratingimmune cells (22). However, the role of these stromal cells inmodulating the immune response in the tumor microenviron-ment, and the influence of the tumor secretome on this capability,is unknown. To test the functional consequences of tumor con-ditioning on the ability of stromal cells to inhibit lymphocyteproliferation and effector function, we used an ex vivo syngeneicculture system, whereby primary Balb/c lymphocytes were cocul-tured with stromal cells conditioned by the tumor secretome inthe presence (MSCTNF-TCM) or absence (MSCTCM) of inflamma-tion [Fig. 1A(i–iv)]. A dose-dependent induction of NF-kB inCT26 in response to TNFa treatmentwas confirmedby a luciferasereporter gene assay (Supplementary Fig. S2A). Primary bonemarrow stromal cells represent a robust model cell type forinvestigation of the effects of tumor conditioning on stromal cellcomponents of the tumor microenvironment, considering theyshare numerous characteristics with intestinal stromal cells in
terms of origin, surface marker expression, gene signatures, andimmunomodulatory capacity (23–25). As previously documen-ted, control MSC had limited ability to reduce CD4þ and CD8þ
T-cell proliferation relative to positive controls [stimulated T cellsand no stromal cells; Fig. 1B(i)]. However, this suppressivecapacity was significantly increased by stromal cell exposure tothe inflammatory tumor secretome [Fig. 1B(ii–iii)]. To identifythe mechanism of stromal cell–mediated suppression, weassessed T-cell viability after coculture. No significant differenceswere observed in either CD4þ or CD8þ T cells cocultured withMSCControl, MSCTCM, or MSCTNF-TCM, suggesting induction ofT-cell anergy or exhaustion rather than activation-induced celldeath (Fig. 1C). In addition to enhanced T-cell suppression,inflammatory tumor-conditioned stromal cells, compared withcontrol or TNFa-primed stromal cells, also significantly reducedlymphocyte activation, measured by TNFa and IFNg release[Fig. 1D(i) and (ii); ref. 26]. A reduction in the cytolytic capacityof lymphocytes was seen when cultured with inflammatorytumor-conditioned stromal cells, measured by a reduction ingranzyme B [Fig. 1D(iii)]. The inhibitory effect exerted bytumor-conditioned stromal cells on T-cell effector phenotype wasmost potent following stromal cell exposure to the secretomefrom TNFa pretreated CT26 cells, indicating a requirement for
Figure 1.
Inflammatory tumor conditioning ofstromal cells results in a significantlyenhanced capacity to inhibit T-cellproliferation and activation. A,Experimental outline. MSC wereseeded in 6-well plates, left to adherefor 24 hours, and subsequently treatedwith 60% tumor-conditioned mediumor controls for 72 hours. Treatmentgroups were as follows: (i) untreated(MSCControl), (ii) TNFa-treated(MSCTNF-a), (iii) CT26-conditionedmedium (MSCTCM), and (iv)conditioned medium from CT26preactivated with TNFa (MSCTNF-TCM).B, (i) Representative histogramsdisplaying CD4þ and CD8þ T-cellsuppression following 72-hourcoculture with tumor-conditionedMSC. Bar charts for (ii) CD4þ and (iii)CD8þ suppression greater than twogenerations. C, Cell viability assessedin CD4þ and CD8þ T cells by flowcytometry after 72-hour coculturewith MSCControl, MSCTCM, MSCTNF-a,or MSCTNF-TCM. SYTOX blue negativitywas used as an indicator of live cells.D,Culture supernatantswere analyzedfor the presence of (i) TNFa (ii) IFNg ,and (iii) granzyme B by ELISAfollowing 72-hour coculture. Errorbars, mean � SEM; � , P < 0.05;�� , P < 0.01; ��� , P < 0.001 by one-wayANOVA and Tukey post hoc test;���� , P < 0.0001; n¼ 3 samples/group,3–5 independent experiments.
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tumor cell inflammatory signaling in dictating stromal cell immu-nomodulatory function. These data demonstrate an enhancedability of stromal cells to prevent the proliferation and effectorfunction of T cells following exposure to the inflammatory tumorsecretome. Considering the close contact between the tumor cellsand the stromal niche in the colon, these data may imply stromalcells utilize the induction of PD-L1 to protect tumor cells fromdestruction by CD8þ cytotoxic T lymphocytes by inhibitingcytotoxicity and inducing T-cell anergy and exhaustion (27).
The inflammatory tumor secretome induces PD-L1 expressionon stromal cells
Stromal cells have been shown to inhibit T-cell responses byboth cell contact–dependentmechanisms, via cell-surface proteinexpression, and cell contact–independent mechanisms, viarelease of soluble mediators (28, 29). Our initial finding of anenhanced immunosuppressive stromal cell potential followingexposure to the inflammatory tumor secretome was observedfollowing contact-dependent interactions between the stromalcells and lymphocytes, and this prompted a comprehensiveanalysis of an array of ligands on the stromal cell surface knownto have immunomodulatory capacity. A significant increase inMHC-I expression following stromal cell exposure to the tumorsecretome was seen, and this effect was significantly enhanced byinflammatory tumor activation [Fig. 2A(i)]. This observationmayhave important consequences, considering tumor antigens can bepresented on MHC-I complexes potentially resulting in dysfunc-tional activation of antigen-specific CD8þ T cells (30). Comparedwith control stromal cells, tumor conditioning, in the presence orabsence of inflammation, had no effect on the surface expressionofMHC-II [Fig. 2A(ii)].CD80expressiondecreased, irrespective ofinflammation, but with expression remaining high [Fig. 2A(iii)],whereas CD86 expression was unchanged [Fig. 2A(iv)].
PD-L1 was found to be specifically increased following tumorconditioning, which was further increased in the presence ofinflammation [Fig. 2B(i); refs. 31, 32]. Expression of PD-L2 andFas Ligand (FasL), two negative regulators of T-cell function, wasfound to be low or almost absent, respectively, and remainedunchanged following exposure to the tumor secretome [Fig. 2B(ii)and (iii); refs. 33–35]. These findings confirmed our previousobservations that T-cell viability remained unchanged followingcoculture. Compared with controls or TNFa-treated stromalcells, where PD-L1 expression was low, inflammatory tumor-conditioned stromal cells expressed PD-L1 to a higher extent thanCT26 tumor cells (Fig. 2C). This finding points to an importantrole for the stromal niche in dictating the fate of tumor-infiltrating, PD-1 receptor–expressing immune cells. Weconfirmed that TNFa, when added to MSCTCM, did not inducestromal cell PD-L1 expression in contrast to MSCTNF-TCM. Thissuggests that TNFa-induced NF-kB signaling in CT26 cells isresponsible for the PD-L1 induction (Fig. 2D). This finding wasfurther confirmed using conditioned media from TNFa-treatedNF-kB–deficient CT26 tumor cells. PD-L1 expression was signif-icantly reduced on MSCTNF-TCM conditioned with CT26/IkB-a SRcells when compared with CT26/EV control cells [SupplementaryFig. S3(i) and (ii)].
Tumor stromal cell suppression of cytolytic CD8þ T cells isreversed by blocking PD-1
To confirm a definitive role for induced stromal cell PD-L1 inthe suppression of T-cell proliferation and activation, we targeted
the PD-1/PD-L1 signaling axis using a monoclonal blockingantibody to the PD-1 receptor.We observed high PD-1 expressionon CD4þ and CD8þ T cells (Fig. 3A) ex vivo. Treatment with anti–PD-1 significantly reduced the ability of inflammatory tumor-conditioned stromal cells to suppress the proliferation of CD8þ Tcells [Fig. 3B(i–ii)]. Following anti–PD-1 treatment, CD8þ T-cellsuppression elicited by stromal cells exposed to the inflammatorytumor secretome was comparable with that induced by stromalcells exposed to the tumor secretome alone or TNFa-treated MSC[Fig. 3B(ii)]. These findings indicated that the enhanced CD8þ
T-cell suppressive capacity of these cells is dependent on stromalPD-L1 expression induced by inflammatory signaling in thetumor microenvironment. No significant differences were seenin the capacity of inflammatory tumor-conditioned stromal cellsto suppress CD4þ T cells, upon treatment with anti–PD-1 [Fig. 3B(iii)]. These findings may indicate a separate unknown mecha-nism by which stromal cells in the tumor microenvironmentmodulate CD4þ T cells.We further confirmed thesefindings usingshRNA CD274 knockdown MSC. shRNA CD274 and scrambleshRNA MSC were extensively characterized for shRNA transduc-tion and knockdown efficiency by microscopy (SupplementaryFig. S4A), mRNA expression (Supplementary Fig. S4B), and flowcytometry (Supplementary Fig. S4C). Quantification of thepercentage of GFPþ shRNA CD274 and scrambled MSC (Supple-mentary Fig. S4D) and of the percentage of PD-L1þMSC from theGFPþpopulation (Supplementary Fig. S4E)were assessed prior tococulture with T cells in vitro. Compared with MSC controls,shRNA PD-L1 MSCTNF-TCM significantly reversed the ability ofMSCTNF-TCM to suppress CD8þ T-cell proliferation [Fig. 3C(i)].Similar to our findings using anti–PD-1, this effect was specificto CD8þ T cells, as the observation was not evident in the CD4þ
T-cell subset [Fig. 3C(ii)]. We also observed a restoration of CD25expression on CD8þ T cells following coculture with shRNACD274MSCTNF-TCM compared withMSCTNF-TCM [Fig. 3D(i)]. TheCD25 restoration was not evident on the CD4þ T cells [Fig. 3D(ii)], validating that the PD-L1–mediated effects were predomi-nantly on theCD8þT cells. PD-1blockadewas sufficient to restorelymphocyte activation and cytolytic potential, measured by IFNg ,TNFa, and granzyme B secretion (Fig. 3E). This demonstrates thecritical role of the PD-1/PD-L1 signaling axis in the ability oftumor-conditioned stromal cells to inhibit CD8þT-cell–mediatedantitumor immune effector functions.
In the context of immunotherapy, biologics targeting PD-L1and PD-1 are showing great promise, but only in a subset ofpatients. A number of clinical trials have relied on measurementsof PD-L1 expression on tumor cells or tumor-infiltrating immunecells in an effort to predict those patients who will respond best,but this method of stratification has had only limited success,with evidence now showing that some patients deemed PD-L1negative will respond to this therapy (19, 36). Our findings mayhighlight an important role for stromal cell PD-L1 expression inmodulating antitumor T-cell responses and in selecting optimalimmunotherapeutic strategies for patients.
T-cell cytotoxicityThe effect of stromal cells on theprogressionof colon cancer has
been tested in vivo, but much of this work has relied on the use ofimmunocompromised mice, limiting the ability to assess theantitumor immune response (37, 38). In order to test the func-tional consequences of stromal PD-L1 expression in vivo, we used
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a fully immunocompetent, syngeneic Balb/c subcutaneoustumor model, whereby the immunogenic CT26 cell line wasadministered with or without control or inflammatory tumor-conditioned stromal cells (39). Because our in vitro analysisshowed that increased PD-L1 expression was unique to stromalcells exposed to the inflammatory tumor secretome, we assessedthis group comparedwith controlMSC in vivo (Fig. 4A). Untreated
MSCwere used as controls as PD-L1 expression on these cells wasnot significantly different to MSCTNF-a or MSCTCM (Fig. 2). Thepreliminary experiments we carried out as part of this studyshowed that control stromal cells induced increased tumorgrowth compared with that of CT26 tumor cells alone (Supple-mentary Fig. S5A), and tumor burden was not different from thatinduced by MSCTNF-a or MSCTCM (Supplementary Fig. S5B). The
Figure 2.
Exposure to the inflammatory tumorsecretome induces markers ofenhanced immunosuppressive abilityon the stromal cell surface. A,Representative histograms and barcharts displaying MFI of MSC, culturedunder the indicated conditions, for (i)MHC-I, (ii) MHC-II, (iii) CD80, and (iv)CD86. B, Histograms and bar chartsdisplaying MFI of MSC, cultured underthe indicated conditions, for (i) PD-L1,(ii) PD-L2, and (iii) FasL. C,Representative histograms and barchart of PD-L1 expression on stromalcells compared with CT26 tumor cells.D, Bar chart showing PD-L1 inductionfollowing addition of TNFa directlyto MSCTCM. Error bars, mean � SEM;� , P < 0.05; �� , P < 0.01; ��� , P < 0.001;���� , P < 0.0001 by one-way ANOVAand Tukey post hoc test; n ¼ 3samples/group, 3–-5 independentexperiments.
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Blocking PD-1 signaling reverses the increased CD8þ T-cell suppression induced by inflammatory tumor-conditioned stromal cells. A, Representative histogramsof PD-1 expression on murine CD4þ and CD8þ T cells isolated from lymph nodes and spleen (n ¼ 3). B, (i) Representative histograms of CD4þ and CD8þ T-cellproliferation with (dark gray/check bars) and without (light gray histogram/black bars) PD-1 blocking antibody in the presence of MSCTNF-TCM. Bar chartsshowing (ii) % CD8þ and (iii) % CD4þ T-cell suppression with and without treatment with PD-1 blocking antibody. C, Bar charts and representative histograms of(i) CD8þ and (ii) CD4þ T-cell proliferation following coculture with CD274 shRNA or scramble shRNAMSC.D,Bar charts showing CD25 expression (MFI) on (i) CD8þ
and (ii) CD4þ T cells following coculture with CD274 shRNA or scramble shRNA MSC. E, Measurement of TNFa, IFNg , and granzyme B by ELISA in culturesupernatants without (black bars) and with anti–PD-1 (striped bars). Error bars, mean � SEM; � , P < 0.05; �� , P < 0.01; ��� , P < 0.00; and ���� , P < 0.0001; byone-way ANOVA or unpaired t test and Tukey post hoc test; n ¼ 3 samples/group, 3–5 independent experiments.
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use of untreated MSC, thereby, allowed us to minimize thenumber of variables tested in our in vivo system.
Evidence exists to suggest that tumor-infiltrating lymphocytescontribute significantly to the prognosis, responsiveness to treat-ment, and likelihood of disease relapse in cancer patients (6).Our analysis of tumor immune cell infiltrates showed no changein the frequency of CD3þ, CD3þCD4þ, or CD3þCD8þ cells [Fig.4B(i–iii); Supplementary Fig. S6]. There was a trend towardreduced frequency of granzyme Bþ CD8þ T cells [Fig. 4B(iv)],but this did not reach statistical significance. Considering the
previously publishedobservation of increased granzymeBexpres-sion in colon tumors correlatingwith favorable patient outcomes,we measured granzyme B expression on CD8þ T cells by medianfluorescent intensity (MFI) and found a significant reduction inthe expression in tumors formed by the coadministration ofCT26 with inflammatory tumor-conditioned stromal cells com-pared with CT26 cells alone. This result was dependent onthe exposure of the stromal population to the inflammatorytumor secretome and, thus, may indicate a role for stromal PD-L1in this effect. Cytotoxic CD8þ T lymphocytes are important in
Figure 4.
Inflammatory tumor-conditionedstromal cells reduce the cytolyticcapacity of tumor-infiltrating CD8þ
T cells resulting in enhanced tumorgrowth and invasiveness. A,Schematic of experimental design.Balb/c mice were injectedsubcutaneously with CT26 cells aloneor in combination with control orinflammatory tumor-conditioned MSCat day 0. Tumors were harvested foranalysis at day 21. B, Tumors wereprocessed to single-cell suspensionsand analyzed by flow cytometry forthe presence of infiltrating (i) CD3þ,(ii) CD4þ, (iii) CD8þ, and (iv)Granzyme Bþ CD8þ lymphocytes. (v)MFI was used to compare granzyme Bexpression on CD8þ T cells betweengroups. � , P < 0.05 by one-wayANOVA and Tukey post hoc test;n ¼ 3–9 mice/group. C, Percentageof animals with an invasive tumorphenotype, defined as any tumor thatpenetrated skin tissue or grew acrossthe peritoneal membrane with tumordeposits found attached to the liverand colon. Error bars, mean � SEM;���� , P < 0.0001 by c2 test for trend(n ¼ 3–9 mice/group). D, Tumorvolume measured by the rationalellipse formula (M1^2 � M2 � p/6).Error bars, mean � SEM; �, P < 0.05;�� , P < 0.01 one-way ANOVA, Tukeypost hoc test. n ¼ 3–9 mice/group,two independent experiments.
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In addition to altering the tumor immune cell infiltrate, stromalcells have been shown to promote the growth and invasion oftumors (41, 42). In our model, injection of CT26 alone inducedsubcutaneous tumor formation with no evidence of tumor cellinvasion and metastasis. In 50% of animals receiving controlstromal cells, tumors were observed to have invaded tissue sur-rounding the primary tumor site and metastasized (Fig. 4C). Thiseffect was significantly potentiated by the coadministration ofstromal cells exposed to the inflammatory tumor secretome(Fig. 4C). In the clinical setting, an invasive or metastatic tumoris associated with a much less favorable outcome for patientsurvival. This holds true in the setting of colorectal cancer, wherethe emergence ofmetastasis is particularly grave (43). A significantincrease in tumor volume was observed upon the coinjection ofstromal cells, with inflammatory tumor-conditioned stromal cellsinducing greater increases in volume than CT26 alone (Fig. 4D).These results highlight a role for inflammatory tumor signaling inaltering the functional characteristics of the tumor stroma. Theobservations of smaller, though significant, increases in invasive-ness and volume upon administration of control stromal cellsmay be indicative of in situ induction of PD-L1 expression oncontrol stromal cells when in contact with tumor cells. Theenhanced increases observed following inflammatory tumor-conditioned stromal cells point to an important role for earlypolarization of the stromal cell phenotype prior to or at thebeginning of tumor formation.
To confirm the immunomodulatory role of inflammatorytumor-conditioned stromal cells and to test the effect of increasingstromal cell number, a second group of animals was coadminis-tered CT26 with double the number of stromal cells used previ-ously (0.5 � 105 vs. 1 � 105). This was investigated using bothcontrol stromal cells and those exposed to the inflammatorytumor secretome. No differences were found in tumor invasive-ness or tumor volumes between animals that had received thelower or higher stromal cell number (Supplementary Fig. S7).Hence, all further experiments and analysis were carried out usingthe lower number of MSC, in consideration of physiologic levelsof MSC in the TME. These data confirmed that the presence of animmunosuppressive stromal niche in a tumor is sufficient tosignificantly affect ability of immune cells to access and eliminatethat tumor. We have shown that the presence of this stromalpopulation caused biologically relevant and potent effects ontumor promotion, even when present in low numbers.
Anti–PD-1 restores CD8þ T-cell activation and reversestumorigenesis
To confirm a role for PD-L1 in the enhanced immunosuppres-sive and tumor-promoting ability of coadministered stromal cellsobserved in vivo, three additional groups of animals were treatedwith anti–PD-1 at 7 and 14 days after tumor induction (Fig. 5A).In line with previous data demonstrating only mild responses toPD-1 monotherapy, in CT26 derived tumors, PD-1 antibodytreatment had no significant effect on the immune cell infiltrateof tumors, and anti–PD-1 treatment also did not alter the fre-quency of CD3þ, CD3þCD4þ, or CD3þCD8þ T cells infiltrating
coinjectedCT26and stromal cell tumors [Fig. 5B(i–iii)].However,a trend toward restoration of the cytolytic capacity of intratumoralCD8þ T cells was seen, measured by granzyme B expression, intumors formed by coadministration of CT26 with inflammatorytumor-conditioned stromal cells [Fig. 5B(iv); refs. 44, 45]. Theimmune cell checkpoint inhibition induced by anti–PD-1 wassufficient to reverse the increased tumor invasiveness that hadbeen observedwith the coadministration of inflammatory tumor-conditioned stromal cells (Fig. 5C). This suggests that stromal cellPD-L1 expressionmay have a central role in enabling the tumor toinhibit CD8þ T-cell–mediated inhibition of metastatic tumorspread. A similar significant reduction in tumor volume wasobserved upon treatment with anti–PD-1 (Fig. 5D). Images oftumors excised from treated animals indicated the increasednumber of invasive nodules on tumors in the CT26 coinjectedwith MSCTNF-TCM group compared with either CT26 alone orCT26 coinjected with MSCControl. This observation of enhancedinvasiveness of tumors was reversed after treatment with anti–PD-1 therapy (Fig. 5E). Tumor volume in animals administeredinflammatory tumor-conditioned stromal cells treated withPD-1 monoclonal antibody was similar to that of animalsadministered CT26 alone. These data point to a central rolefor stromal cell PD-L1 expression in obstructing the activity ofantitumor CD8þ immune responder cells, allowing tumors togrow through immune evasion.
PD-L1 is differentially expressed on cancerous stroma inclinical samples
Because our experiments to date had been carried out usingmurine tissue, we next wanted to confirm the phenomenon ofstromal cell PD-L1 induction on stromal tissue in the humantumor microenvironment. We examined transcriptional profilesof colorectal cancer resection samples (data setGSE35602),whichhad been laser-capture microdissected (LCM) to isolate the stro-mal and epithelial fractions prior to microarray profiling (20).Assessment of PD-L1 gene expression (CD274) indicated signif-icantly higher expression of PD-L1 in the stromal compartment ofCRCs compared with the epithelial cells [Fig. 6A(i)]. Assessmentof EpCam (EPCAM), E-caderhin (CDH1), as markers of epitheliallineage, and a-SMA (ACTA2) and vimentin (VIM), as markers ofmesenchymal lineages, further confirmed the purity of the sam-ples following LCM [Fig. 6A(ii)]. Assessment of PD-L1, PDGFR-a(PDGFRA), CD90 (THY1), andCD105 (ENG) indicated that geneexpression of these markers was associated with the stromalcompartment in colorectal cancer tissue samples. Because of thisobservation, we next investigated if exposure to the tumor secre-tome would induce human stromal cell PD-L1 expression in amanner similar to that seen for our murine cells. Using primaryhumanbonemarrow stromal cells fromhealthydonors,whichweshowed were CD90, CD105, and PD-L1–positive [Fig. 6B(i–iii)],and the secretome frommicrosatellite (HCT116) or chromosom-al (HT29) unstable cells, we repeated the conditioning protocoloutlined in Fig. 1A (46). We observed a significant increase inPD-L1 expression upon exposure to the inflammatory tumorsecretome, and this induction occurred irrespective of tumor cellmicrosatellite instability (HCT116) or chromosomal instability[HT29; Fig. 6B(iii–v); ref. 46].
Ourdata inbothmouse andhumanmodels have indicated thatPD-L1 expression is elevated in MSC following exposure toconditioned media from inflammatory stimulated colon cancercells. Cancer-associated fibroblasts (CAF) represent a major
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Treatment with an anti–PD-1 restoredantitumor immune responses resulting indecreased invasiveness and tumor growth.A, Schematic of experimental design. Balb/cmice were administered CT26 cells alone or incombination with control or inflammatorytumor-conditioned MSC at day 0. Anti–PD-1was administered i.p. on days 7 and 14.Tumors were harvested for analysis at day 21.B, Tumors were processed to single-cellsuspension and analyzed by flow cytometryfor the presence of infiltrating (i) CD3þ, (ii)CD4þ, and (iii) CD8þ T cells. (iv) MFI was usedto compare granzyme B expression on CD8þ
T cells between groups. One-way ANOVAand Tukey post hoc test. C, Percentage ofanimals with an invasive tumor phenotype(defined in 4C). Error bars, mean � SEM;� , P < 0.05; ���� , P < 0.0001 by Fisher exacttest. D, Tumor volume measured by therational ellipse formula (M1^2 � M2 � p/6).Error bars, mean � SEM; �, P < 0.05 byone-way ANOVA and Tukey post hoc test;E, Representative images of tumor tissuedissected from animals at day 21 with (right)or without (left) anti-PD-1. n¼ 3–9 per group.n ¼ 2 independent experiments.
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component of colorectal cancer stroma and provide a suitablemodel to test our findings in colorectal cancer clinical samples.Therefore, we utilized gene-expression profiles (data setGSE70468) derived from patient-matched primary fibroblasts(n ¼ 14 samples from 7 patients) isolated from within colorectalcancer tissue (CAFs), to represent MSC following cancer cellexposure, or from adjacent, distant, normal mucosal tissue (nor-mal), to represent unconditioned MSC (20). Analysis of overall
gene-expression profiles indicated a nonsignificant trend (P 0.10;range, 1.15–1.08-fold change) toward increased PD-L1 geneexpression in CAFs compared with normal fibroblasts(Fig. 6C). A pairwise analysis of patient-matched samples furtherconfirmed this finding, with 6 of the 7 matched CAFs displayingan increase in PD-L1 gene expression comparedwith their patient-matched normal fibroblast (Supplementary Fig. S8). This analysisin two sets of independent colorectal cancer clinical tissue cohorts
Figure 6.
PD-L1 (CD274) expression isdifferentially expressed on cancerousstroma in clinical samples and is alsoinduced on human stromal cellsexposed to the inflammatory tumorsecretome. A, (i) Relative CD274(PD-L1) gene-expression profile onepithelial and stromal cells fromcolorectal cancer patients (n¼ 13; dataset GSE35602). (ii) Clustering forgene-expression profiles of PDGFR-a(PDGFRA), PD-L1 (CD274), CD90(THY1), CD105 (ENG), Vimentin (VIM),a-SMA (ACTA2), E-cadherin (CDH1),andEPCAM (EPCAM).B,Healthydonorhuman stromal cells expression of (i)CD90, (ii) CD105, and (iii) PD-L1.Primary human MSC were treated withconditioned medium from control orTNFa-activated (iv) HCT116 or (v)HT29 human colon tumor cells andPD-L1 expression (MFI) was measuredby flow cytometry. C, Relative PD-L1gene expression in normal and CAFsfrom colorectal cancer patients (n¼ 7;data set GSE70468). Error bars,min–max; �� , P < 0.01; ��� , P < 0.001;���� , P < 0.0001 by unpaired t test.
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supports our observations in the mouse model and adds furtherweight to our suggestion of an important role for stromal cell PD-L1 induction in colorectal cancer.
DiscussionIn this article, we described a central role for stromal cell PD-L1
expression in the tumormicroenvironment in aiding the tumor inavoiding immune cell–mediated clearance. This dampening ofthe antitumor immune responses by the stromal compartmentresulted in enhanced tumor burden and invasiveness (Fig. 7). Thestromal compartment is located adjacent to both the cancerousepithelial cells and the colonic vasculature and lymphatic net-work, representing a physical barrier to entry for immune cells inresponse to inflammation or epithelial transformation (47). Inaddition to this structural role, these cells have been shown toproduce solublemediators and express proteins that can influenceimmune and epithelial cells in diseased tissue (48). We have nowshown that PD-L1, induced in response to activation of inflam-matory signaling in the tumor, is a criticalmediator of stromal cellimmunomodulatory capacity. More specifically, we identified
tumor cell NF-kB as an important signaling pathway in theinduction of a tumor-promoting stromal cell phenotype,demonstrated by a lack of induced stromal cell PD-L1 expressionin the absence of TNFa-induced NF-kB activation in CT26 cells.Given the inflammatory nature of the colon tumor microenvi-ronment and the important role for NF-kB signaling in thepathogenesis of colon cancer, these results may highlight amechanism by which NF-kB signaling in colon tumor cellsenables communication with the adjacent stromal compartment,thereby dictating their immunomodulatory function (49, 50).Interestingly, a study by Lakins and colleagues has shownthat CAFs have similar properties as those described here, interms of their ability to suppress antitumor responses. However,the effects highlighted in their study were shown to be dependenton T-cell death mediated by FasL and PD-L2. In contrast, wehighlighted a significant role for tumor-induced stromal cellPD-L1 in cell death–independent suppression of CD8þ T-cellproliferation and activation, indicating induction of anergy.These results may highlight the importance of the nature of thetumormicroenvironment on the induction of stromal cell–medi-ated immunosuppression (i.e., the presence of inflammation or
Figure 7.
TNFa-induced colon cancer signaling induces stromal cell PD-L1, which inhibits cytotoxic CD8þ T-cell antitumor immune responses and promotes colon cancer.Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by theinhibition of activated CD8þ granzyme B-secreting T cells in vitro, and the inhibition of CD8þ effector cells was associated with enhanced tumor progression.Targeting the PD-L1/PD-1 signaling axis reversed the MSC PD-L1–induced immunosuppression and tumor-promoting effects.
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hypoxia). Increasing our understanding of the impact of stromalcell functional status, as well as other tumor-associated immu-nosuppressive cell populations in different tumor microenviron-ments, will be essential to improving the efficacy of immu-notherapies in the future.
By utilizing an immunocompetent, syngeneic murinemodel, we could overcome the limitations in assessingimmune cell infiltrate associated with the use of xenograftsin immunocompromised mice. This allowed us to identifyCD8þ T-cell granzyme B as a potential effector moleculeinhibited by the stromal cell barrier in the tumor microenvi-ronment, which was restored by anti–PD-1 therapy. Withspecific regard to colon cancer, higher intratumoral expressionof granzyme B has been shown to correlate with improvedsurvival in patients (51). Collectively, these data demonstratethe importance of the stromal compartment in modulatingthe CD8þ T-cell–mediated antitumor immune responses in aPD-L1–dependent manner.
Expression of PD-L1 has been primarily detected on the surfaceof epithelial neoplastic cells in a number of cancers. However, incolorectal carcinoma, IHC-based studies of small cohorts havedetected high PD-L1 expression in the stromal and immunecompartments (18, 52, 53). In light of studies showing positiveresponses to PD-1 immunotherapy in patients whose tumorshave been deemed PD-L1–negative, we provide a rationale forthe assessment of stromal cell PD-L1 expression in order to betterstratify patients for immunotherapy (19).
We also showed the phenomenon of inflammatory tumor-induced PD-L1 expression on human stromal cells in response tothe secretome from microsatellite- or chromosomal instablehuman colon tumor cells. This is particularly important in lightof data showing a better response to PD-1 immunotherapy inMSI-high colon cancer patients. We suggest that this is, as wasdiscussed by Huang and Wu, a result of a lower mutational loadattractingmuch fewer tumor-infiltrating CD8þ T cells (54). In thissetting, PD-1 inhibition is of limited use because a paucity ofT cells, whose effector function can be disinhibited by suchtherapy, exists. However, as the authors suggest, treatment witha second immune-stimulatory agent, such as MEKi, could lead toCD8þ T-cell infiltration and, thus, synergistic and favorableresponses in these patients (54, 55). We validated our in vitroand in vivo findings in two independent colorectal cancer clinicalresection cohorts. Using transcriptional profiles specific for thestromal or epithelial fractions of overall colorectal cancer tissue(20), we identified significantly higher gene expression for PD-L1in the stroma, and we also identified increased PD-L1 geneexpression inCAFs comparedwithpatient-matchednormal colonfibroblasts.
The precise molecular mechanisms underpinning thisenhanced PD-L1 expression on stromal cells remain to be eluci-dated and represent an important avenue of pursuit for the futurein our laboratory. Identificationof the factors released from tumorcells under conditions of inflammation, and the signaling path-ways activated in MSC, may facilitate the development of noveladjuvants for immunotherapy to enhance clinical therapeuticeffects. In summary, targeting stromal cell PD-L1 may be key tobreaking the cycle of immune evasion and immunosuppressionestablished by the stromal compartment of the tumor microen-vironment, leading to more favorable and durable outcomes forpatients.
Disclosure of Potential Conflicts of InterestA.E. Ryan reports receiving commercial research support from Janssen
Pharmaceuticals and Bristol-Myers Squibb and has ownership interest inU.S. Patent app 15/798,670. No potential conflicts of interest were disclosedby the other authors.
Authors' ContributionsConception and design: T. Ritter, L.J. Egan, A.E. RyanDevelopment of methodology: G. O'Malley, K. Lynch, S.D. Naicker, A.E. RyanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. O'Malley, O. Treacy, K. Lynch, S.D. Naicker,N.A. Leonard, P. Lohan, A.E. RyanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. O'Malley, K. Lynch, S.D. Naicker, P.D. Dunne,T. Ritter, L.J. Egan, A.E. RyanWriting, review, and/or revision of the manuscript: G. O'Malley, K. Lynch,T. Ritter, L.J. Egan, A.E. RyanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. O'Malley, O. Treacy, K. Lynch, S.D. Naicker,N.A. Leonard, P.D. Dunne, A.E. RyanStudy supervision: A.E. Ryan
AcknowledgmentsThe authors would like to acknowledge financial support from the Irish
Cancer Society (CRF12RYA) and Science Foundation Ireland (15/SIRG/3456)andGalway University Foundation toDr. A.E. Ryan, a postgraduate scholarshipfrom the Irish Research Council (GOIPG/2013/998) to GO'Malley, and a grantfrom Science Foundation Ireland (12/IA/1624) to T. Ritter.
The authors thank Professor Matthew Griffin, Professor Rhodri Ceredig, andDr. Declan McKernan for helpful discussions; Ms. Georgina Shaw, Dr. JoanaCabral, and Ms. Athina Rigalou for technical assistance; and Dr. Shirley Hanleyin the NUIG Flow Cytometry core facility for technical expertise.
The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received October 20, 2017; revised June 14, 2018; accepted September 11,2018; published first September 18, 2018.
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T-cell Antitumor Immune Responses andPromotes Colon CancerIn the original version of this article (1), the paper had missing information in thelegend for Fig. 5B and 5D and had aminor error in the representative image in Fig.6B.The errors have been corrected in the latest online HTML and PDF versions of thearticle. The authors sincerely regret these unintentional omissions.
In the original version (1) for Fig. 5B (i-iii, v) and 5D, the samples used as controls hasbeen clarified. The new information is contained in the figure's legend. Thesecorrections do not alter the main conclusions drawn from this study.
Figure 5. Treatment with an anti–PD-1 restored antitumor immune responsesresulting in decreased invasiveness and tumor growth. A, Schematic of experi-mental design. Balb/c mice were administered CT26 cells alone or in combinationwith control or inflammatory tumor-conditioned MSCs at day 0. Anti–PD-1 wasadministered i.p. ondays 7 and 14. Tumorswere harvested for analysis at day 21.B,Tumors were processed to single cell suspension and analyzed by flow cytometryfor thepresence of infiltrating (i)CD3þ, (ii)CD4þ, and (iii)CD8þT cells. ForCT26controls, MSCControl and MSCTNF-TCM from Fig. 4B (i-iii) were used. (iv) Medianfluorescent intensity (MFI) was used to compare granzyme B expression onCD8+ Tcells between groups. One-way ANOVA and Tukey's post hoc test. C, Percentage ofanimals with an invasive tumor phenotype (defined in 4C). Error bars: mean �SEM; �,P<0.05; ����,P<0.0001by Fisher exact test.D,Tumor volumemeasuredbythe rational ellipse formula (M1^2�M2� p/6). For CT26 controls, MSCControl andMSCTNF-TCM fromFig. 4B (v)were used. Error bars:mean� SEM; �, P < 0.05 by one-way ANOVA and Tukey's post hoc test; E, Representative images of tumor tissuedissected from animals at day 21 (right) with or (left) without anti–PD-1. n = 3-9per group. n = 2 independent experiments.
In the original version (1) for Fig. 6B, the experimental controls, the number ofhealthy donor samples used, and number of independent experiments performedwas clarified, and labeling of the panels was corrected. For Fig. 6B (iv, v), therepresentative histogram for MSCTCM in (iv) and (v) was mistakenly the same curvedue to a drag-and-drop error in the analysis program used and has been corrected inthe new figure. These corrections do not alter the quantitative data and mainconclusions drawn from this study.
Figure 6. PD-L1 (CD274) expression is differentially expressed on cancerousstroma in clinical samples and is also induced on human stromal cells exposedto the inflammatory tumor secretome. A, (i) Relative CD274 (PD-L1) geneexpression profile on epithelial and stromal cells from CRC patients (n = 13;Dataset GSE35602). (ii) Clustering for gene expression profiles of PDGFR-a(PDGFRA), PD-L1 (CD274), CD90 (THY1), CD105 (ENG), Vimentin (VIM),aSMA (ACTA2), E-cadherin (CDH1) and EPCAM (EPCAM). B, Healthy donorhuman stromal cells expression of (i) CD90, (ii) CD105, and (iii) PD-L1. PrimaryhumanMSCs (n=1donor)were treatedwith conditionedmedium fromcontrol orTNFa-activated (iv) HCT116 or (v) HT29 human colon tumor cells, and PD-L1expression (MFI) was measured by flow cytometry (n = 3 independent experi-ments). Error bars: mean � SEM, P < 0.05 by one-way ANOVA and Tukey post hoctest. ��, P < 0.01; ���, P < 0.001; ����, P < 0.0001 by unpaired t test.C,Relative PD-L1gene expression innormal and cancer-associatedfibroblasts fromCRCpatients (n=7; Dataset GSE70468). Error bars: Min-Max; Unpaired t test.
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Published online January 2, 2019.doi: 10.1158/2326-6066.CIR-18-0889�2019 American Association for Cancer Research.
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