Immunologic and Metabolic Features of Pancreatic Ductal ......Immunologic and Metabolic Features of Pancreatic Ductal Adenocarcinoma Deﬁne Prognostic Subtypes of Disease Jack Hutcheson1,

Biology of Human Tumors

Immunologic and Metabolic Features ofPancreatic Ductal Adenocarcinoma DefinePrognostic Subtypes of DiseaseJack Hutcheson1, Uthra Balaji1, Matthew R. Porembka2,3, Megan B.Wachsmann4,Peter A. McCue5, Erik S. Knudsen1,2, and Agnieszka K.Witkiewicz1,2,4

Abstract

Purpose: Pancreatic ductal adenocarcinoma (PDA) is associ-ated with an immunosuppressive microenvironment that sup-ports the growth of the malignancy as well as immune systemevasion. Here we examine markers of immunosuppression inPDA within the context of the glycolytic tumor microenviron-ment, their interrelationship with tumor biology and associationwith overall survival.

Experimental Design: We utilized tissue microarrays consist-ing of 223 PDA patients annotated for clinical stage, tumor size,lymph node involvement, and survival. Expression of CD163,FoxP3, PD-L1, and MCT4 was assessed by IHC and statisticalassociations were evaluated by univariate and multivariate anal-ysis. Multimarker subtypes were defined by random forest anal-ysis. Mechanistic interactions were evaluated using PDA cell linesand models for myeloid differentiation.

Results: PDA exhibits discrete expression of CD163,FoxP3, and PD-L1 with modest individual significance. How-

ever, combined low expression of these markers was associ-ated with improved prognosis (P ¼ 0.02). PDA tumor cellsaltered macrophage phenotype and function, which sup-ported enhanced invasiveness in cell-based models. Lactateefflux mediated by MCT4 was associated with, and requiredfor, the selective conversion of myeloid cells. Correspond-ingly, MCT4 expression correlated with immune markers inPDA cases, and increased the significance of prognostic sub-types (P ¼ 0.002).

Conclusions: There exists a complex interplay between PDAtumor cells and the host immune system wherein immuno-suppression is associated with negative outcome. MCT4expression, representative of the glycolytic state of PDA, con-tributes to the phenotypic conversion of myeloid cells. Thus,metabolic status of PDA tumors is an important determinant ofthe immunosuppressive environment. Clin Cancer Res; 22(14);3606–17. �2016 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDA) is a highly aggressive

malignancy and the fourth leading cause of cancermortality in theUnited States.Most patients presentwith advanced disease and theprognosis of these patients is dismal, with 5-year overall survivalless than 6% and a median survival of 4 to 6 months (1). Evenwhen complete surgical resection is possible, recurrence is com-mon and a majority of patients will die with distant metastasiswithin 3 years (2). As systemic treatments remain minimallyeffective, many studies have focused on the unique genetic char-acteristics of PDA todefinenew therapeutic targets (3).Meanwhile,

recent studies have demonstrated unique aspects of metabolismand immune evasion in PDA that could offer rational targets fortherapeutic intervention beyond genomic targets (1, 4–6).

PDA is characterized by the presence of desmoplastic stromathat can account for asmuch as 90%of the total tumor volume (7,8). Interactions between the tumor cells and the surroundingstroma create an inflammatory microenvironment that is condu-cive to tumor growth and progression. This chronic inflammationrecruits predominantly T cells supported by additional leuko-cytes, including dendritic cells, macrophages, neutrophils, and Bcells, to the primary tumor (7, 9). The clinical relevance of thisongoing inflammatory program is reflected in poor outcomescorrelated with infiltrating inflammatory cells and the contribu-tions of specific cell types are being increasingly elucidated (10,11). For instance, neutrophil-lymphocyte ratio may represent aprognosticmarker in PDA patients with a lower ratio representingimproved prognosis and, despite the lack of a significant increasein the total cell number, B cells have now been implicated in PDAprogression through their ability to impact myeloid cell function(12–14). Meanwhile, single-agent inhibitors of immune check-points have not demonstrated substantial efficacy in PDA (15).Thus, a better understanding of the mechanisms that regulate thePDA immunosuppressive environment could reveal specific dis-ease subtypeswith enhancedprognostic significance andallow foradditional targeted treatment approaches.

Glycolysis plays an important role in inflammation and, specif-ically, lactate is a criticalmediator of themacrophage inflammatory

1McDermott Center for Human Growth and Development, UT South-western Medical Center, Dallas, Texas. 2Simmons Cancer Center, UTSouthwestern Medical Center, Dallas, Texas. 3Department of Surgery,UT Southwestern Medical Center, Dallas, Texas. 4Department ofPathology, UT Southwestern Medical Center, Dallas, Texas. 5Depart-ment of Pathology, Thomas Jefferson University, Philadelphia,Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Agnieszka K. Witkiewicz, UT Southwestern MedicalCenter, Dallas, TX 75390-8591. Phone: 214-648-3111, ext. 84159; Fax: 214-648-1666; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1883

�2016 American Association for Cancer Research.

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response (16–18). Alterations in glycolytic tumormetabolism alsoimpact the contributions of other immune cells in PDA (19, 20).Since increased expression of the lactate exporterMCT4 (SLC16A3)also portends poor outcome in PDA (4), we hypothesized that theglycolytic imbalance in pancreatic cancer cells, represented byMCT4expression, directly impacts the surrounding immunemilieuand that these characteristics could be used to identify distinctdisease subtypes. To this end, in this study we examine the tumormicroenvironment of PDA patients for indicators of an immuno-suppressive phenotype and utilize these elements to define prog-nostic subtypes of disease. Furthermore, we demonstrate in vitrothat the metabolic context of pancreatic cancer cells can influencethe phenotype of associated immune cells and, by extension,expression of the lactate exporter MCT4 can further refine thedisease-immune subtypes.

Materials and MethodsTumor microarray construction and population study

Study cases were obtained from the surgical pathology files atThomas Jefferson University with Institutional Review Boardapproval. The tissue microarray (TMA) contained tumor samplesderived from 223 largely consecutive patients with PDA who hadbeen treated at Thomas Jefferson University Hospitals betweenthe years 2002 and 2010. Whole tissue section slides were con-structed and TMAs were derived from them using a tissue arrayer(Veridiam) as described previously (4). IHC was performed asdescribed previously (21) on 4-mmTMA sections using a standardavidin–biotin immunoperoxidase method with antibodies spe-cific for CD163 (1:100, clone 10D6, Leica), FOXP3 (1:50, clone206D, Biolegend), PD-L1 (CD274, 1:200, clone E1L3N, CellSignaling Technology), and MCT4 [1:250, developed and char-acterized by Dr. Nancy Philp (22)]. Staining was performed on aVentana Benchmark automated stainer.

TMA analysisAfter staining, positive cells were counted and converted to a

percentage of the total counted cells in a givenfield and expressionof all examined markers was categorized as low or high based on

either the median percentage of positive cells or 10% positivity(PD-L1), consistent with published reports (5, 23, 24). Correla-tion between markers was obtained using the spearman correla-tionmethod. Kaplan–Meier (KM) curves for both independent aswell as combined markers were obtained using the survivalpackage in R statistical software (25). Statistical significance andHR along with 95% confidence intervals for the KM curves wasestablished using the log-rank P value obtained from Cox pro-portional hazard regression analysis. An unsupervised randomforest (RF) clustering method was used for clustering analysis ofimmunemarkers (26). RF dissimilarity measure betweenmarkerswas obtained and classified into clusters using the partitioningaroundmedoidsmethod; both implemented using RF and clusterpackages in R. All heatmaps and three-dimensional scatterplotswere obtained using heatmap.2 and rgl packages in R.

Immune infiltrate scoringLymphocytes and neutrophils were assessed in hematoxylin

and eosin–stained whole tissue sections at 10� magnification ineither the central area of the tumor (tumor cellular environmentfield) or at the tumor's invasive margin (tumor stroma field). Thepresence of infiltrate was assessed using a four-degree scale in eacharea, where a score of 0 indicated no presence, 1 denoted a mildand patchy appearance of inflammatory infiltrate (rare), 2 signi-fied a prominent inflammatory reaction (intermittent), and 3represented dense infiltration (frequent).

CellsEstablished pancreatic cancer cell lines (BXPC3, Capan-2,

Hs766t, MIA PaCa-2, Panc-1, PL5, PL45) were cultured in DMEMþ10% FBS. THP-1 cells were a generous gift fromDr. David Farrarand were maintained in RPMI1640 supplemented with 10% FBS,L-glutamine (2 mmol/L), sodium pyruvate (1 mmol/L), nones-sential amino acids, and b-mercaptoethanol (75 mmol/L). Tovisualize wound healing, PL45 cells were stably transfected withmCherry fluorescent protein. ForMCT4 knockdown experiments,a mixture of Lipofectamine RNAiMAX transfection reagent (LifeTechnologies) and MCT4 siRNA (h2, sc-45892, Santa Cruz Bio-technology) was added to plates of 70% confluent pancreaticcancer cells as described by the manufacturer.

Macrophage differentiation and polarizationFor macrophage differentiation assays, THP-1 cells were cul-

tured for 72 hours in the presence of 50 ng/mL phorbol 12-myristate 13-acetate (PMA) or 50% conditioned media, as indi-cated. For polarization assays, following incubation in the pres-ence of PMA, adherent THP-1 cells were washed and differenti-ation media was replaced with fresh supplemented RPMI with orwithout 50% conditioned media. Conditioned media was iso-lated from approximately 80% to 90% confluent establishedpancreatic cancer cell lines 72 hours postsplit, or posttransfection,centrifuged at 4,000 rpm for 10minutes, and filtered with a 0.45-mm syringe filter to remove contaminating cells and debris beforeuse. For proteinase K and lactic acid experiments, followingconditionedmedia collection, samples were treated with protein-ase K (50 mg/mL) for 30 minutes at 37�C followed by phenyl-methylsulfonyl fluoride (PMSF, 5 mmol/L) for 1 hour at roomtemperature to inactivate proteinaseK. Lactic acid (SigmaAldrich)was added at 8 mmol/L. Lactate readings were acquired on an

Translational Relevance

Recentfindings in pancreatic ductal adenocarcinoma (PDA)have shown that an influx of immunosuppressive inflamma-tory cells around the tumor is associated with poor outcome.However, additional insights are necessary to determinewhether these prognostic findings can be utilized as predictivemarkers. We used a cohort of PDA patients to characterize theimmunosuppressive tumor microenvironment and demon-strated the association of multiple markers with clinical out-come. Utilizing established PDA cell lines, we showed that by-products of glycolysis are capable of skewing immune cellphenotypes. On the basis of these findings, we refined ourimmune expression panel by incorporating the lactate export-erMCT4. Patientswith lowexpressionof immunemarkers andMCT4 exhibited a significantly improved overall survival.Thus, metabolic features of PDA can impact the local immuneenvironment, which is likely relevant to prognosis and treat-ment of disease.

Immunologic and Metabolic Features can Define PDA Prognosis

www.aacrjournals.org Clin Cancer Res; 22(14) July 15, 2016 3607




automated electrochemical analyzer (Bioprofile Basic-4 Analyzer,NOVA Biomedical).

Flow cytometryAdherent THP-1 cellswere scraped fromculture dishes, collected

by centrifugation, and resuspended in flowwash buffer (PBSþ1%FBS þ 0.1% sodium azide). To stain for cell surface markers, cellswere incubated in the presence of antibodies specific for galectin 3/Mac-2 (LGALS3, clone M3/38, Cedarlane Laboratories), CCR7/CD197 (clone 3D12), CD206 (MRC1, clone 19.2), and HLA-DR(clone L243; eBioscience). Cells were then washed and resus-pended in 1% paraformaldehyde. Pancreatic cancer cells werecollected by trypsinization and resuspended as described above.For MCT4 staining, intracellular staining was performed asdescribed previously (27) using anti-MCT4 antibody (cloneH-90, Santa Cruz Biotechnology) conjugated with Alexa fluor555 (Z-25305, Life Technologies). Following incubation, cellswere washed with 0.1% saponin and then resuspended in flowwash buffer. All sampleswere collected on an LSRII flow cytometer(BD Biosciences) and analyzed using FlowJo software (FlowJo).

Drug sensitivityCells from established pancreatic cancer cell lines were plated

for 72 hours in the presence or absence of PMA-differentiatedmacrophages, pancreatic cancer cell–conditioned media–polar-ized macrophages, and gemcitabine (90 nmol/L). At the end ofthe time course, relative cell number was determined by CellTiter-Glo cell viability assay (Promega) and luminescencewas read on aSynergy 2 multimode plate reader (Biotek Instruments).

Cancer cell invasivenessTHP-1 cells were plated in 12-well plates, differentiated for 72

hours with PMA, and then polarized with conditioned mediaovernight. The following day, fresh media was added and trans-well membrane inserts with 0.4-um pores (Corning) were addedonto each well. Pancreatic cancer cells (10,000 cells/membrane)were added to the top of each transwell membrane and culturedfor 24 hours. The top of each insert was carefully washedwith PBSand the remaining cells on the bottom of the membrane werefixed in 10% formalin and stainedwith hematoxylin. Membraneswere cut from the transwell supports, placed onmicroscope slides,and imaged. The number of migrated cells was counted and theaverage number of cells was obtained from 10 high-poweredfields. For wound-healing assays, equivalent numbers of eitherPMA-differentiated or conditioned media-polarized THP-1 cellswere added to wells of culture dishes confluent with pancreaticcancer cells. Each well was scored with a pipet tip and imaged atthe indicated time points at 40� total magnification on an EVOSFL Cell Imaging System (Life Technologies).

ResultsDifferential recruitment of immune cells to tumor and stromalcompartments is associated with survival differences in PDApatients

Pancreatic ductal adenocarcinoma often arises in the back-ground of chronic inflammation suggesting that perturbationsin immune function can have significant impact related to tumorbiology, disease prognosis, and therapeutic interventions. Toelucidate the association of suppressive immune features inclinical cases a cohort of PDA cases (n ¼ 223) was employed

(Table 1). Within this cohort, inflammation, as defined by thepresence of lymphocytes and neutrophils, was widely distributedthroughout the cases (Supplementary Fig. S1A) and overall lym-phocyte or neutrophil accumulation was not significantly asso-ciated with survival (Supplementary Fig. S1B).

Proper T-cell function is necessary for efficient recognition andsubsequent elimination of tumor cells by the immune system. Assuch, suppression of T-cell function is associated with pooroutcome in multiple malignancies, including pancreatic cancer(28). Under normal circumstances, FoxP3þregulatory T cells playa critical role in blunting the T-cell response to prevent autoim-munity; however, the role of these cells is subverted in the contextof tumorigenesis and disease progression (29, 30). As PDA oftencontains abundant desmoplastic stroma, regulatory T cells werescored in the tumor cellular environment and tumor stroma(stroma). Approximately 50% of PDAs harbored FoxP3-positiveinfiltrate scored as high in at least one compartment (Fig. 1A).Higher levels of these cells only in the stroma were associated(HR ¼ 1.395, P < 0.05) with worse survival and combinedexpression in both compartments was associated (HR ¼ 2.038,P < 0.05)with decreased survival versus the absence of FoxP3 (Fig.1B). These data suggest that the immune milieu of the surround-ing stroma is an important determinant of disease outcome inaddition to that of the tumor itself (Supplementary Table S1).

The presence of M2 (CD163þ) macrophages was evaluated inboth the tumor cellular environment and stroma (Fig. 1C).Expression of CD163 corresponded (HR ¼ 2.058, P¼ 0.005) tooverall survival when present in the tumor cellular environment,as opposed to stromal expression that only trended (HR¼ 1.481,P < 0.08) toward poor survival (Fig. 1D). Likewise, the combinedabsence of CD163 throughout both the tumor and stroma wassuggestive (HR ¼ 1.852, P < 0.08) of a favorable prognosis(Fig. 1D; Supplementary Table S2). Patients with tumors expres-sing low levels of both FoxP3 and CD163 had a significantlyimproved outcome [median survival (MS) ¼ 45.6 months]when compared against patients with high levels of both markers(HR ¼ 2.367, P > 0.017, MS ¼ 21 months). Meanwhile, highCD163 expression in combination with low FoxP3 expression

Table 1. Patient characteristics

Characteristics No. of patients 223 (%)

Median age (range) 65 (27–89)GenderMale 121 (54)Female 102 (46)

Tumor size (cm)0–2 42 (19)2.1–4 123 (55)>4 49 (22)Unknown 9 (4)

Node involvementPositive 142 (64)Negative 78 (35)Unknown 3 (1)

Grade1 20 (9)2 138 (62)3 57 (26)Unknown 8 (3)

Vital statusAlive 119 (53)Dead 92 (42)Unknown 12 (5)

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Clin Cancer Res; 22(14) July 15, 2016 Clinical Cancer Research3608




Figure 1.Immunosuppressive cells are associated with pooroutcome in PDA. A, representative images of highFoxP3 staining in the tumor cellular environment (TCE,>0%) and stroma (>9.5%) of patient sections. B,KM plots indicating survival probability in humanpatients with high or low accumulation of FoxP3þcellsin the tumor cellular environment and/or stroma. SeeSupplementary Tables S1 and S2. C, representativeimages of High CD163 staining in the tumor cellularenvironment (>30%) and stroma (>33%) of patientsections. D, KM plots indicating survival probability inhuman patients with high or low accumulation ofCD163þcells in the tumor cellular environment and/or stroma. See Supplementary Tables S3 and S4. E,KM plots indicating survival probability in humanpatients with high or low accumulation of CD163þandFoxP3þcells in the tumor stroma. See SupplementaryTable S5.






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was significantly associated (HR ¼ 3.422, P < 0.005, MS ¼ 13.8months) with the shortest median survival (Fig. 1E; Supplemen-tary Table S3).

Pancreatic cancer cells influencemacrophage differentiation ina cell line–dependent manner

To further characterize the relationship between pancreaticcancer cells and macrophages, THP-1 cells were stimulated withconditioned media from various pancreatic cancer cell lines(Fig. 2A). Although the conditionedmedia demonstratedmarkedincreases in macrophage differentiation over media alone, theresponse varied greatly between the different cell lines in terms ofmorphology (Fig. 2B) as well as overall adherence (Fig. 2C).Phenotypically, these differences were characterized by theincreased prevalence of a galectin-3 (Mac-2)–negative populationinMIA PaCa-2 or PL45 conditionedmedia–treated THP-1 cells ascompared with those cells treated with Capan-2 conditionedmedia (Fig. 2D). Furthermore, these galectin-3–negative cellsexpressed markers indicative of both M1 (CCR7, HLA-DR) andM2 (CD206) polarized macrophages (Fig. 2E and F).

Macrophages differentiated by pancreatic cancer cells enhancecancer cell invasiveness

Given that PDA cell–conditioned media differentially inducedmacrophage differentiation and activation, the impact thesemacrophages played onmechanisms of pancreatic cancer severitywas gauged. Regardless of the initial stimuli, treated THP-1 cellshad no discernable effect on gemcitabine response (Fig. 2G).However, the addition of the macrophages resulted in some celldeath in the cocultures, as evidenced by decreased cellularity inTHP-1-only groups. Conditioned media-differentiated macro-phages promoted pancreatic cancer cell invasiveness, asmeasuredby Boyden chamber (Fig. 2H) and wound-healing assays (Fig. 2Iand J). Of note, expedited wound healing is a cell line–specificphenomenon, as evidenced by the differential impact PMA-dif-ferentiatedmacrophages have on Capan-2 cells as compared withthe other cell types.

Combined expressionof immunosuppressivemarkers defines asubset of PDA patients with improved prognosis

The presence of alternatively activated, M2 macrophages andregulatory T cells in the PDA tumors suggested local immuneevasion, whereby tumor cells escape immune surveillance. There-fore, the expression of PD-L1, which is associated with suppres-sion of local immune engagement, was evaluated in the PDAcohort. Tumor-specific expression of PD-L1 was observed in themajority of PDA cases (Fig. 3A) and was associated (HR ¼ 1.619,P¼ 0.056) with overall survival (Fig. 3B) suggesting that the

presence of PD-L1 could be important for the maintenance ofimmune evasion.

Tumors that were dually absent of FoxP3 and PD-L1 (Fig. 3C),or CD163 and PD-L1 (Fig. 3D) demonstrated prolonged overallsurvival. In all patients, the median survival was 21 months,whereas these dual negative cases had amedian survival of greaterthan 45 months (Supplementary Tables S5 and S6). Importantly,this was not a feature of all combinations of these markers, asanalysis of CD163- or FoxP3-negative stroma combined with theabsence of PD-L1 expression had no such survival advantage.These data suggested that interactions between the immunemarkers could be clinically significant and define a subset of PDApatients with favorable prognosis.

RF analysis was used to cluster cases based on the presence ofCD163, PD-L1, and FoxP3 (Fig. 3E). Using this approach, fourclusters of cases thathaddistinct principle component featuresweredefined(Fig.3F). Inparticular, cluster3,consistingofapproximately25%ofcases inthecohort,was largelydevoidofCD163,FoxP3, andPD-L1, and had improved overall survival that was statisticallysignificant versus clusters 1 and2and trended for improved survivalversus cluster 4 (Supplementary Table S7). In comparison with allotherclusters, the cases in cluster3were significantlyassociatedwithimproved outcome exhibiting a median survival in excess of 50months (Fig. 3G; Supplementary Table S8).

Pancreatic cancer cell–mediated changes in glycolyticmetabolites influencemacrophage differentiation independentof cytokines

Local factors alter the function of immune cells in the tumormicroenvironment. Established PDA cell lines secrete varyingamounts of cytokines that would impact immune function,including high expression of CSF-1 by MIA PaCa-2 cells (31).To determine whether the differences in macrophage activationwere attributable to variations in cytokine secretion, THP-1cells were cultured in protein-depleted conditioned media.Although protein depletion caused a significant reduction inmacrophage adherence (Fig. 4A), the phenotypic differencesremained (Fig. 4B). These data suggest that although secretedproteins (e.g., cytokines and chemokines) are important med-iators of THP-1 adherence, additional factors are sufficient toskew macrophage polarization.

Glycolysis has also been identified as a regulator of the inflam-matory response (32). The PDAmicroenvironment is glycolytic incomparisonwith surrounding tissue andhas been associatedwithpoor outcome in PDA (4). As such, we investigated how theglycolytic environment impacts immune cell activation in PDA.Similar to our previous report (4), various established PDA celllines differentially express MCT4 (Fig. 4C), which serves to export

Figure 2.PDA cell products influence the phenotype and function ofmyeloid cells. A, relative level of THP-1 cell adherence induced by the indicated conditionedmedia (n¼ 3).B, representative phase contrast images (20�) of THP-1 cells treated with PMA or the indicated conditioned media for 72 hours. Arrows denote cellularmorphology indicative of activatedmacrophages. Scale bars represent 100 mm. C, the relative number of adherent THP-1 cells following treatment with the indicatedconditioned media for 72 hours (n ¼ 3). D, representative pseudocolor dot plots depicting the differential expression of galectin-3 in THP-1 cells treated with theindicated conditioned media for 72 hours (n¼ 3). E, the relative number of Gal3-CCR7þCD206þand Gal3þCCR7þCD206þmacrophage populations in THP-1cells treatedwith PMAor the indicated conditionedmedia for 72 hours (n¼ 3). F, representative histogramoverlay (left) and quantitative analysis (right) of THP-1 cellsurface expression of HLA-DR following 72 hours treatmentwith the indicated conditionedmedia. G, relative cell survival following treatment of PDA cell lines (n¼ 3)with the indicated combination of gemcitabine (GEM) and/or THP-1 cells differentiated in PMA or PDA cell–conditioned media (PaCa CM). H, the number of PDAcells from the indicated cell lines (n¼ 3) that migrated to the bottom of a transwell membrane in response to PDA-conditioned media activated THP-1 cells.Data were collected from 10 randomly selected high-power fields (40�) per sample. I, representative images of wound healing in mCherry-expressing PL45 cellsafter 48-hour incubation with or without conditioned media (CM) differentiated THP-1 cells. J, wound healing in the indicated PDA cell lines over the courseof 48 hours� THP-1 cells predifferentiated for 72 hours with either PMA or matched PDA CM. For all panels, all data are representative of at least three experiments.Graphed data represent mean values � SEM. � , P < 0.05; �� , P < 0.01.






increased levels of lactate produced via glycolysis to the extracel-lular environment. Accordingly, the cell lines with increasedMCT4 expression consumemore glucose and secretemore lactatethan those with lower MCT4 expression (Fig. 4D). Meanwhile,the addition of exogenous lactic acid to conditioned medianegated the phenotypic differences induced by the different PDAcell lines (Fig. 4E). To determine whether MCT4 expression onPDA cells influenced immune response, MCT4 was knockeddown in PDA cell lines (Fig. 4F) resulting in a significant decreasein lactate secretion and glucose consumption in PDA cell lineswith higher basal MCT4 expression (Fig. 4G). Conditionedmedia from these cells was added to undifferentiated THP-1cells and in all cases, MCT4 knockdown resulted in decreasedTHP-1 cell differentiation, as measured by macrophage adher-ence, when compared with control cells (Fig. 4H). Although theaddition of lactic acid did not increase THP-1 adherence in these

cells following MCT4 knockdown (Fig. 4H), this treatment wassufficient to rescue CCR7 and CD206 expression (Fig. 4I). Thesedata are consistent with our earlier findings (Fig. 4Aand B) insuggesting differential roles for glycolytic metabolites in adher-ence and polarization.

The inclusion of MCT4 expression enhances the prognosticvalue of immunosuppressive biomarkers

Given the role of MCT4 in regulating immune function, addi-tional prognostic power could be added by incorporating immu-nologic markers withMCT4 expression. Thus, we investigated therelationship ofMCT4 expression in PDAwith the immune-relatedmarkers. These data showed that MCT4 status in both the tumorcellular environment and stroma were positively correlated withCD163 expression in the tumor cellular environment (Fig. 5A).

Figure 3.Immune evasion and suppressionsignature correlates with poorprognosis in PDA. A, representativeimages of low (<10%) and high (�10%)PD-L1 staining of patient PDA tumorsections. B, KM plots indicating survivalprobability in human patients with highor low accumulation of PD-L1þcells. SeeSupplementary Table S6 and S7. C, KMplots indicating survival probability inhuman patients with high or lowaccumulation of FoxP3þand PD-L1þcells in the tumor cellularenvironment (TCE). SeeSupplementary Table S8. D, KM plotsindicating survival probability in humanpatients with high or low accumulationof CD163þand PD-L1þcells in the tumorcellular environment. SeeSupplementary Table S9. E, heat mapsof unsupervised RF clustering ofimmune marker expression. Clusterswere defined using the partitioningaround medoids method. F, KM plotsindicating survival probability in humanpatients based on the previouslydefined immune marker expressioncluster. See Supplementary Table S10.G, KM plots indicating survivalprobability in human patients from thecluster of lowest complex expression(cluster 3)with those from clusterswithhigh expression of at least one marker(clusters 1, 2, 4). See SupplementaryTable S11.

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All other markers exhibited a tenuous or nonsignificant associa-tion with MCT4 in the tumor cellular environment, but interest-ingly stromal MCT4 correlated with veritably all immunologicfeatures (Fig. 5A). These findings suggest that although there is arelationship between the glycolytic state of the tumor and theimmunologic features of disease, the dual absence of both isassociated with increased overall survival. RF clustering revealedthe presence of discrete clusters that were separated on the basis of

distinct principle component features (Fig. 5B). Cluster 4 lackedalmost all immune markers and the expression of MCT4 in thestroma. In KM analysis, this cluster harbored particularly favor-able overall survival (Fig. 5C; Supplementary Table S9). Impor-tantly, this cluster was significant (HR¼ 3.852, P < 0.005,MS > 53months) versus all other cases (MS¼ 20.6months) and remainedsignificant in multivariate analysis including grade and lymphnode status (Fig. 5D; Supplementary Tables S10 and S11).

Figure 4.Pancreatic cancer cell metabolism influences myeloid cell differentiation and phenotype. A, relative number of adherent THP-1 cells (n¼ 3 per group) following24-hour treatment with the indicated conditionedmedia� proteinase K. B, representative expression of CCR7 and CD206 on THP-1 cells (n¼ 3 per group) followingexposure to the indicated proteinase K pretreated conditioned media. C, representative expression of MCT4 by the indicated pancreatic cancer cell lines. D,amount of lactate secreted and amount of glucose consumed per 1 � 106cells by the indicated pancreatic cancer cell lines (n¼ 3 per group). E, representativeexpression of CCR7 and CD206 on THP-1 cells (n ¼ 3 per group) following exposure to the indicated lactic acid pretreated conditioned media. F, representativeimmunoblots of MCT4 expression in PDA cell lines � siMCT4. G, amount of lactate secreted and amount of glucose consumed per 1 � 106cells by the indicatedpancreatic cancer cell lines � siMCT4 knockdown (n¼ 3 per group). H, relative level of THP-1 cell adherence induced by conditioned media collected fromthe indicated PDA cell lines� siMCT4 knockdown and� lactic acid (n¼ 3). I, representative expression of CCR7 and CD206 on THP-1 cells (n¼ 3 per group) followingexposure to MIA PaCa-2 conditioned media under the indicated conditions. For all panels, all data are representative of at least three experiments. Grapheddata represent mean values � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.005.






Discussion

On the basis of underlying tumor metabolism and mutationstatus, PDA actively promotes an inflammatory and immuno-suppressive microenvironment by recruiting macrophages andother immune cells. The composition of the cellular immunemicroenvironment in PDA is associated with variable patientoutcome. We examined the tumor microenvironment of patientswith PDA and identified tumor characteristics that were indepen-dently associated with prognosis. In addition, PDA can influencemacrophages recruited to the tumor through alterations in themetabolic environment. MCT4 expression, a surrogate marker forthe glycolytic tumor microenvironment, influenced macrophagedifferentiation in vitroand provided additional prognostic dis-crimination in patients.

The immune system is designed to recognize and removepathologic insults, including malignancy, with the survival ofcancer cells suggests a host immunedeficiency or an active evasionby cancer cells. To this end, the makeup of the immune milieu inthe PDA tumormicroenvironment and the effects these cells haveon pathogenesis and prognosis has garnered increased attention.Since cytotoxic (CD8þ) T cells are abundant in PDA (14), the lackof tumor cell death would seem to be the result of an immuno-suppressive microenvironment instead of an intrinsic defect ofadaptive immunity. This is further supported by immunotherapy

models where targeting the T-cell checkpoint PD-L1 improved theefficacy of systemic therapy (23). However, the use of immunecheckpoint blockade therapeutics as single-agent treatments hasresulted in only minimal success (15, 33, 34), suggesting thatsome additional aspects of PDA or the tumor microenvironmentare promoting immunosuppression.

The combined blockade of both myeloid differentiation sig-naling (CSF1R) and T-cell checkpoint signaling (PD-L1) has beenshown to be more effective than either approach individually(35). Although these data suggest that myeloid cell–mediatedpromotion of immunosuppression directly contributes to thegeneral lack of efficacy of single-agent immunotherapeutics inPDA, the mechanisms that promote this phenomenon remainunclear. Thus, although the emergence of this suppressiveimmune environment supporting PDA development providesnew possibilities to distinguish disease subtypes and highlightsnew opportunities for therapeutic interventions, additionalinsights are necessary to guide these beneficial outcomes.

PDA is associated with a significant desmoplastic stroma thatharbors infiltrating immune cells. Previously, increased tumorinfiltrate of FoxP3-, CD163-, or PD-L1-expressing cells or M2macrophages has been associatedwith tumor progression or pooroutcome (5, 24, 36, 37). We expand on this finding by demon-strating the importance of the tumor stroma to the developmentof this suppressive phenotype. PDA stroma has been shown to

Figure 5.Suppressive immune features and MCT4 expression define prognostic disease subtypes in PDA. A, Pearson correlation heat maps of immune markers and MCT4expression. B, heat maps of unsupervised RF clustering of immune marker and MCT4 expression. C, KM plots indicating survival probability in human patientsbased on the previously defined immune marker and MCT4 expression clusters. See Supplementary Table S12. D, KM plots indicating survival probability in humanpatients from the cluster of lowest complex expression (cluster 4) with those from clusters with high expression of at least one marker (clusters 1, 2, 3, 5).See Supplementary Tables S13 and S14.

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support the recruitment and accumulation of immune cells,demonstrating a phenotype high in proinflammatory cytokineand chemokine expression (7). RF clustering of our suppressiveimmune phenotypes reflects the association between suppressiveimmune cells in the stroma and inferior outcomes (clusters 1 and2). Although the exact mechanism is not clear, the stroma in PDApromotes primary tumor growth by creating an immunosuppres-sive microenvironment. Further examination of the relationshipbetween the primary tumor and the stroma is important to betterunderstand the biology of this aggressive cancer and tohelp devisenovel therapies.

It has recently been demonstrated that macrophages are criticalfor PDA tumor growth and that KRAS status plays an importantrole in macrophage recruitment and activation (38–40). Toexplore this further, here we utilized a system by which cells fromthe monocytic THP-1 line were treated with conditioned mediaisolated from established PDA cell lines in vitro. Our data dem-onstrate that these PDA cell lines differentially influence macro-phage development. For example, the phenotype induced byconditioned media isolated from MIA PaCa-2 and PL45 cells(CCR7þCD206þ) strongly resembles that of myeloid-derivedsuppressor cells (MDSC), which would further enhance theimmunosuppressive environment (41). These cells also expressthe increased HLA-DR expression as compared with cells differ-entiated by Capan-2 conditioned media. It has previously beenshown that the expression of HLA-DR on MDSCs is associatedwith their ability to induce CD4þT-cell tolerance, further reducingthe capability for an effective antitumor immune response (42).However, HLA-DR expression on these cells also provides amechanism by which specifically designed CD4þT cells can con-vert these MDSCs, decreasing their suppressive efficacy (42). Thissuggests that PDA influences the immune tumor microenviron-ment by modulating macrophage populations.

Tumor-associated macrophages have been shown to promotetumor growth through various mechanisms including TLR4/IL10–mediated epithelial-mesenchymal transition, promigratoryfactor secretion, and enhanced drug resistance (37, 43–46). Here,we show that macrophages increase invasiveness in establishedPDA cell lines, as demonstrated by increased recruitment ofPDA cells (Fig. 2H) and expedited wound-healing response(Fig. 2I and J) in the presence of macrophages differentiated byPDA-conditionedmedia. MIA PaCa-2 and PL45 cells were able toovercome the effect of PMA-differentiated macrophages onwound healing whereas Capan-2 cells were not. Thus, our datasuggest a PDA cell–specific mechanism that facilitates tumorgrowth and prevents antitumor response, in line with previousreports (38, 39, 47). Taken together, these data highlight thecomplex regulation of the immune environment in PDA.

Although it has been shown that tumor-derived cytokines suchas GM-CSF can directly influence the immune milieu in andaround the tumor (38, 39), our data indicate that a cytokine-independent mechanism also contributes to skew immune celldifferentiation (Fig. 4B). PDA can directly influencemacrophages,myeloid cells, and the tumor microenvironment by altering thelocal metabolic conditions. It has become increasingly evidentthat glycolysis plays an important role in regulating immuneresponse, particularly where increased glycolysis produces glyco-lytic by-products like lactic acid (32). In macrophages, high levelsof intracellular lactate result in decreased responsiveness to LPS,and thus macrophage MCT4 expression is associated with more

effective inflammatory response (16). Conversely, tumor-derivedlactic acid is capable of skewing macrophages toward the anti-inflammatory, tumor-promoting M2 phenotype (48). Here weshowdecreasedmacrophage differentiation in THP-1 cells treatedwith conditioned media from cell lines where MCT4 has beenknocked down. These data provided sufficient rationale for exam-ining how PDA expression of MCT4 is associated with immunemarkers of disease.

Extensive profiling of established PDA cell lines has revealeddistinct stratification of these lines on the basis of their metaboliccharacteristics. These findings demonstrate that various cell linesare fundamentally predisposed toward different metabolic pro-grams, including a subset of cells with an elevated reliance onglycolysis (49). Along these lines, we have recently shown thatenhanced glycolysis in PDA, represented by the expression ofMCT4, is associated with prognosis. Our findings demonstratedthat glycolysis was largely suppressed in PDA cell lineswith higherendogenous levels ofMCT4 (e.g., PL45 andMIA PaCa-2) whereascell lines with lower endogenousMCT4 expression were generallyunaffected (4). Here we have demonstrated that MCT4 knock-down is sufficient to attenuate lactate efflux and reduce glucoseuptake (Fig. 4G), consistent with our previous findings, whichalso indicated that MCT4 is necessary to maintain proper lactateproduction (4). Strikingly, patients that had low expression ofMCT4 in conjunction with diminished markers of suppressiveimmune cells demonstrated significantly improved survival ascompared with other clusters. These data suggest that a com-bination of immune and metabolic features in PDA definesubsets of disease that correspond to prognosis. Taken together,these data suggest that the individual metabolic state of PDAmalignancies may portend the efficacy of potential immu-notherapies and that, ultimately, a combined approach com-prised of immunotherapy and a targeted metabolic therapymay increase treatment efficacy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Hutcheson, M.R. Porembka, P.A. McCue, E.S.Knudsen, A.K. WitkiewiczDevelopment of methodology: J. Hutcheson, A.K. WitkiewiczAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Hutcheson, M.B Wachsmann, P.A. McCue,A.K. WitkiewiczAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Hutcheson, M.R. Porembka, M.B Wachsmann,E.S. Knudsen, A.K. WitkiewiczWriting, review, and/or revision of the manuscript: J. Hutcheson, U. Balaji,M.R. Porembka, M.B Wachsmann, E.S. Knudsen, A.K. WitkiewiczAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.S. KnudsenStudy supervision: A.K. Witkiewicz

Grant SupportThis work was supported by grants from NIH.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisementinaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 4, 2015; revised January 7, 2016; accepted January 25, 2016;published OnlineFirst February 8, 2016.






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2016;22:3606-3617. Published OnlineFirst February 8, 2016.Clin Cancer Res Jack Hutcheson, Uthra Balaji, Matthew R. Porembka, et al. Adenocarcinoma Define Prognostic Subtypes of DiseaseImmunologic and Metabolic Features of Pancreatic Ductal

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Immunologic and Metabolic Features of Pancreatic Ductal ......Immunologic and Metabolic Features of Pancreatic Ductal Adenocarcinoma Deﬁne Prognostic Subtypes of Disease Jack Hutcheson1,