Epithelial Mesenchymal Transition in Human Prostate Cancer ...Cancer Res; 78(16); 4671–9. 2018 AACR. Introduction Prostate cancer is a heterogeneous disease in terms of patho-logic

Tumor Biology and Immunology

Epithelial–Mesenchymal Transition in HumanProstate Cancer Demonstrates Enhanced ImmuneEvasion Marked by IDO1 ExpressionKimberley Kolijn1, Esther I. Verhoef1, Marcel Smid2, Ren�e B€ottcher3,Guido W. Jenster3, Reno Debets2, and Geert J.L.H. van Leenders1

Abstract

Cancer invasion and metastasis are driven by epithelial–mesenchymal transition (EMT), yet the exact mechanismsthat account for EMT in clinical prostate cancer are notfully understood. Expression of N-cadherin is considered ahallmark of EMT in clinical prostate cancer. In this study,we determined the molecular mechanisms associated withN-cadherin expression in patients with prostate cancer. Weperformed laser capture microdissection of matched N-cadherin–positive and -negative prostate cancer areas frompatient samples (n ¼ 8), followed by RNA sequencing. N-cadherin expression was significantly associated with animmune-regulatory signature including profound upregu-lation of indoleamine 2,3-dioxygenase (IDO1; log2-foldchange ¼ 5.1; P ¼ 2.98E-04). Fluorescent immunostain-ings of patient samples confirmed expression of IDO1protein and also its metabolite kynurenine in primarily

N-cadherin–positive areas. N-cadherin–positive areas alsoexhibited a local decrease of intraepithelial cytotoxic(CD8þ) T cells and an increase of immunosuppressiveregulatory T cells (CD4þ/FOXP3þ). In conclusion, EMT inclinical prostate cancer is accompanied by upregulatedexpression of IDO1 and an increased numberof regulatory T cells. These data indicate that EMT, whichis an important step in tumor progression, can be pro-tected from effective immune control in patients withprostate cancer.

Significance: These findings demonstrate EMT is linkedto an immunosuppressive environment in clinical pro-state cancer, suggesting that patients with prostate cancercan potentially benefit from combinatorial drug therapy.Cancer Res; 78(16); 4671–9. �2018 AACR.

IntroductionProstate cancer is a heterogeneous disease in terms of patho-

logic growth patterns, molecular aberrations, and clinical out-come. While prostate cancer is currently graded according to theGleason scoring system, analysis of individual growth patternsprovides additional information on tumor cell biology andclinical behavior (1). We recently found that Gleason score 7prostate cancer with "ill-formed" architecture was enriched fortumor cells that express N-cadherin, and represents a morpho-logic substrate for epithelial–mesenchymal transition (EMT) inpatients with prostate cancer (2).

EMT is characterized by the acquisition of a mesenchymal-likephenotype by epithelial cells, and mediates tumor invasion andmetastasis (3). Epithelial cells that undergo EMT in vitro becomespindle shaped and upregulate mesenchymal markers such as

vimentin, fibronectin, N-, and OB-cadherin combined with sup-pression of epithelialmarkers such as keratins and E-cadherin (4).

N-cadherin has been recognized as a robust marker forEMT both in vitro and in vivo (5–8). Although expression ofN-cadherin is rare in localized low-grade prostate cancer, it isincreased in high-grade progressive disease (9–11). Concom-itant N-cadherin upregulation and E-cadherin downregulation,also referred to as cadherin switching, was found to be the mostreliable marker for EMT in prostate cancer patients' samples(2). While various mechanisms involved in cadherin switchingand EMT have been identified in vitro, the underlying regulatorypathways in clinical prostate cancer are unclear. Therefore, theobjective of this study was to determine the molecular andcellular mechanisms associated with N-cadherin expression inpatients with prostate cancer by using microdissection and RNAsequencing.

Materials and MethodsPatient specimens

Patients with prostate cancer, who had undergone surgicalremoval of the prostate (radical prostatectomy) without receivingprior radiation or hormonal therapy for their disease, were select-ed at the Erasmus Medical Center (Rotterdam, the Netherlands;Supplementary Table S1). Prostate specimens were transportedon ice after surgery to the pathology department. A transversetissue slide was snap frozen in liquid nitrogen for researchpurposes and stored at�196�C until use. The remaining prostatewas injected with neutral-buffered formalin (4%) to ensure fast

1Department of Pathology, Erasmus Medical Center, Rotterdam, the Nether-lands. 2Department of Medical Oncology, Erasmus Medical Center, Rotterdam,the Netherlands. 3Department of Urology, Erasmus Medical Center, Rotterdam,the Netherlands.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Kimberley Kolijn, Department of Pathology, ErasmusMedical Center, P.O. Box 2040, 3000 CA, Rotterdam, the Netherlands. Phone:316-5000-1696; Fax: 31-10-7038340; E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-17-3752

�2018 American Association for Cancer Research.

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and equal fixation prior to paraffin embedding. All prostatespecimens were evaluated by a urogenital pathologist (GvL) whorecorded Gleason score according to the WHO/ISUP 2014 guide-lines, as well as pT-stage (WHO 2009), surgical margin status andtumor growth pattern (1). The use of residual tissue for scientificpurposes was approved by the institutional Medical ResearchEthics Committee (MEC-2011-295 and MEC-2011-296). Sam-ples were used in accordance with the "World Medical Associa-tion-Declaration of Taipei on Ethical Considerations regardingHealth Databases and Biobanks," as well as national and inter-national guidelines. Informed consent was obtained through anopt-out procedure, as described in the "Human Tissue and Med-ical Research: Code of conduct for responsible use" developed bythe Dutch Federation of Medical Scientific Societies (FMWV,version 2002, update 2011).

ImmunostainingsSlices of 4- to 5-mm were cut from formalin-fixed, paraffin-

embedded tissues or frozen tissue and mounted on Starfrost(Knittel) silane-coated glass slides for IHC or KP Frost (Klinipath,theNetherlands) glass slides forfluorescent immunostaining (IF).For IHC, paraffin-embedded tissues were deparaffinized withxylene and rehydrated. Endogenous peroxidase activity wasblocked with 0.3% hydrogen peroxide in PBS for 20 minutes.Heat antigen retrieval was performed with a specialized micro-wave (MicroMed T/T Mega) with tissues merged in citrate buffer(pH 6.0, Sigma-Aldrich) for 15 minutes. Slides were incubatedovernight at 4�C with primary antibodies diluted in PBS thatcontained 2% BSA. Secondary antibodies were incubated for 30minutes at room temperature and visualized using the EnVisionsystem (Dako). All primary and secondary antibodies are spec-ified (Supplementary Table S2).

For immunostainings on frozen sections, slides were fixated informalin (4%) and permeabilized with 0.5% TritonX-100 in PBSfor 10 minutes. Slides were washed with PBS, blocked with 10%BSA/PBS and incubatedwithprimary antibody in 2%BSA/PBS for1 hour at room temperature or at 4�C overnight. After washingwith PBS, slides were incubated with secondary antibodies andchromogenically visualized (EnVision Dako Kit, Dako). For fluo-rescent immunostainings, slides were incubated with secondaryantibodies labeled with Alexa488 and Alexa594. Slides weremounted in Vectashield (Vector Laboratories) containingHoechst 34580 (4,000�, Life Technologies) to visualize nuclei.Immunofluorescent tissue sections were analyzed using a confo-cal laser scanning microscope (Zeiss LSM 700, Carl Zeiss) withZEN 2 imaging software (Carl Zeiss).

To quantify regulatory T cells (CD4þ/FOXP3þ) in N-cadherin–positive and -negative regions, we performed immunofluorescenttriple staining (n ¼ 3). Sections were incubated with primaryantibodies overnight, washed with PBS-T and PBS, followed byincubation with secondary antibodies labeled with Alexa647 andAlexa594. After washing, an additional incubation step for 1 hourat room temperature with CD4 conjugated to Alexa 488 wasperformed. The number of CD4þ/FOXP3þ cells was scoredwithin an N-cadherin–positive or -negative area between 2.2 and3.2 mm2. The regulatory T cell count was corrected for area size. Toquantify lymphocytic infiltrates, we performed chromogenic dou-ble staining of CD4 or CD8 with N-cadherin using the VentanaBenchmark Ultra platform (Ventana Medical Systems; n ¼ 10).Ultraview universal DAB and Alkalin phosphatase were used forthe detection and chromogenic staining of secondary antibodies.

The number of CD4þ and CD8þ cells was scored using photo-graphs made using a light microscope, and a small open areaoverlay in Photoshop (Adobe CS6, Adobe Systems Incorporated)to randomly select scoring areas and mask other tissue areas.

Laser capture microdissection and RNA isolationWe performed hematoxylin/eosin staining and N-cadherin

IHC on frozen tissue samples derived from radical prostatectomyspecimens containing >20% tumor cells. The specimens werepreselected after screening a larger number of frozen prostatesamples for the presence of ill-formed Gleason grade 4 and N-cadherin expression (Supplementary Table S1). Eight samplesthat contained tumor areas with more than a hundred N-cad-herin–positive cells were selected for laser capture microdissec-tion (LCM) and subsequent RNA sequencing. N-cadherin–posi-tive and -negative areas were identified by matching both ill--formed architecture and N-cadherin expression in consecutivereference slices. Every two slices, an additional reference slice wascut for IHC staining to identify and confirm N-cadherin–positivetissue regions in LCM slices. N-cadherin–positive and -negativeprostate cancer areas (�0.15 mm2) present in the same tissuesection were captured separately in LCM tubes (AdhesiveCap500opaque, Carl Zeiss; Supplementary Fig. S1). These areas containedboth malignant epithelial cells and adjacent stroma.

LCM and RNA isolation were performed as described previ-ously (12). Cresyl Violet acetate (Sigma-Aldrich) was used to stainslides and visualize morphology. RNA was isolated using themiRNeasy Micro Kit (Qiagen) according to the manufacturer'sguidelines and included an on-column DNAse digestion step(RNase-Free DNase set, Qiagen). RNA Integrity Number (RIN)and quantity were measured in duplicate in a bioanalyzer (2100Bioanalyzer, Agilent Technologies) using the RNA 6000 PicoKit (Agilent Technologies) according to manufacturer's protocol.The mean quality of total RNA isolated by LCM was 26.1 ng(median 20.6; range 8.5–63.3 ng) with RIN values between6.1 and8.4 (mean7.3;median7.4). TheRNAquality andquantitywas not significantly different between N-cadherin–positiveand –negative regions.

RNA preamplification, sequencing, and data workflowRNA preamplification and sequencing were performed by

AROS (AROS Applied Biotechnology A/S). Total RNA was pre-amplified (Ovation RNAseq v2 System NuGen.) and used togenerate double stranded cDNA from mRNA and nonpolyade-nylated transcripts. The cDNA libraries were prepared from allsamples and one control sample (high-quality human referenceRNA) according to the KAPA Library Preparation Kits (Illuminaseries, KK8200 and KK8201). Samples were fragmented to 300–500 base pair libraries with an ultrasonicator (M22 Focused,Covaris) and DNA quality was measured (High Sensitivity DNA,Agilent Technologies). High-quality samples were then paired-end sequenced with TruSeq SBS V4 chemistry on the HiSeq2500(Illumina). An average of 111.2million (total) reads wasmappedper sample (Supplementary Table S3). All RNA sequence datawere aligned to the preindexed UCSC human reference genome19with TopHat version 2.0.4 and expression levels quantified viaHTSeq-count (version 0.5.4p1; ref. 13). Accuracywas increased byalignment against the indexed transcriptome prior to alignmentto the genome. Subsequently, the edgeR package (version 3.0.4)was used to investigate differentially expressed genes betweenN-cadherin–positive versus -negative prostate cancer areas (14).

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Differentially expressed genes with a P < 0.05 and a log2 foldchange (log2FC) smaller or larger than 1 were analyzed (equal tohalve or double gene expression), and functional analyses wereperformed with Ingenuity Pathway Analysis (IPA, Qiagen).

Cell lines and qPCRProstate cancer cells lines, LNCaP, PC3, and DU145, were

obtained from the ATCC (15). All cell lines were maintained at37�C, 5% CO2 in RPMI medium supplied with 5% FCS andpenicillin/streptomycin (Lonza). Authentication was mostrecently performed using STR analysis in the year 2014 andMycoplasma testing was done on a yearly basis with theMycoAlert Mycoplasma Detection Kit (Lonza). Cells were pas-saged for a maximum of 6 months for the experiments done inthis study after thawing. After 24-hour culture in serum-deplet-ed DMEM medium without phenol-red (11039–021, Lonza),cells were stimulated in 4-fold with 100 mmol/L L-Kynurenine(K8625, Merck) in the same medium. Subsequently, RNAwas isolated using the RNeasy Kit (Qiagen, Venlo) accordingto manufacturer's protocol. Total RNA was reverse transcribedto cDNA using oligo dT12 primers and M-MLV reverse tran-scriptase (Invitrogen). Real-time PCR reactions contained 5 mL20� diluted cDNA in Taqman Universal PCR Master Mix,AmpliTaq, and 1� FAM primer/probe mix (CDH1, Entrez GeneID 999; CDH2, Gene ID 1000; and IDO1, Gene ID 3620) in atotal of 25 mL. Amplified products were quantified relative tothe geomean of hydroxymethylbilane synthase (HMBS,HS00609293_G1) and glyceraldehyde-3-phosphate dehydro-genase (GAPDH, HS02758991_G1; Applied Biosystems). All Ctvalues were quantified using the 7500 Real-Time PCR system(Applied Biosystems) with a threshold at 0.2 within the expo-nential curve.

Statistical analysisAll statistics were performed using SPSS 20 (SPSS) orGraphPad

Prism (GraphPad Software, Inc). Data were tested for normalitywith the Shapiro–Wilk test. A Mann–Whitney U test was per-formed to compare the number of CD4þ and CD8þ cells inimmunohistochemically stained slices and gene expression inprostate cancer cell lines (PC3, LNCaP, and DU145). A two-sidedP < 0.05 was considered significant.

ResultsN-cadherin expression is associated with alteredimmunoregulatory pathways

RNA sequencing of paired N-cadherin–positive and negativeprostate cancer regions revealed 871differentially expressed genes(P� 0.05; Supplementary Table S4). Ingenuity Pathway Analysis(IPA) of genes with at least 2-fold change (�1� log2FC �1) inexpression (n ¼ 668) revealed that the majority of genes wereassociated with cancer (86.5%), organismal injuries/abnormali-ties (87.0%), growth and proliferation (40.4%), cell movement

Figure 1.

Heatmap of differential expression of inflammatory response genes inN-cadherin–positive clinical prostate cancer. Heatmap diagram of differentiallyexpressed, inflammatory response genes in N-cadherin–positiveversus –negative clinical prostate cancer samples. RNA was isolated fromN-cadherin–positive (Ncad pos) and –negative (Ncad neg) microdissectedtumor areas. Gene expression data were obtained using the Illumina HiSeq2500platform, TopHat, and edgeR. Differentially expressed geneswith a P < 0.05 and�1 1 were analyzed with IPA. The diagram represents73 differentially expressed inflammatory response genes identified by IPA,

which were centralized around the median and clustered. Columns representhierarchical clustered prostate cancer samples and N-cadherin–positivesamples are shown (bold italic). Each row represents an inflammatory responsegene, while expression levels are shown by log2-fold change and colorsindicate up- (red) or downregulation (green). The heatmap demonstratesthat inflammatory response genes are differentially expressed inN-cadherin–positive versus –negative prostate cancer samples.

EMT and Immune Evasion in Clinical Prostate Cancer

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(24.7%), andmorphology (22.6%; Supplementary Table S5).Weidentified 25 differentially expressed genes associated with EMT(as defined by IPA), including OB-cadherin (CDH11; log2FC ¼1.1; P ¼ 0.02), TGFB1 (log2FC ¼ 1.3; P ¼ 0.02) and Wnt familymember 5A (WNT5A; log2FC ¼ 1.8; P ¼ 0.01; SupplementaryTable S6). Overexpression of N-cadherin at mRNA level (CDH2;log2FC ¼ 2.6; P ¼ 5.82E-04) confirmed the accuracy of LCM. Wedid not detect significantly different expression of other EMT-associated genes, such as fibronectin (FN1) and vimentin (VIM).In addition, transcription factors SNAIL, SLUG, TWIST, ZEB1, andZEB2 that have all been associated with EMT induction in vitrowere not differentially expressed in N-cadherin–positive prostatecancer regions (16–21).

Various differentially expressed genes were linked to immunecell trafficking (33.4%) and inflammatory response (17.4%),such as CD28 signaling in T helper cells (15 genes), iCOSsignaling in Thelper cells (14 genes), and IL4 signaling (12 genes).

In total, IPA identified 73 inflammatory response genes that weredifferentially expressed in N-cadherin–negative versus -positivesamples (Fig. 1). In addition, the IPA upstream regulator featureidentified IFNg , TGFB1 and lipopolysaccharide as potential dri-vers based on the observed gene expression changes in the dataset.Immunosuppressive genes such as indoleamine 2,3-dioxygenase 1(IDO1; log2FC ¼ 5.1; P ¼ 2,98E-04), tryptophan-2,3-dioxygenase 2(TDO2, log2FC ¼ 6.6, P ¼ 4,68E-03), cytotoxic T-lymphocyte-asso-ciated protein 4 (CTLA4, log2FC ¼ 2.9, P ¼ 0.01), and T-cellimmunoreceptor with Ig and ITIM domains (TIGIT; log2FC ¼ 4.9;P¼ 1,59E-04)were overexpressed inN-cadherin–positive regions(Supplementary Table S4). Both IDO1 and TDO2 are enzymesinvolved in the degradation of tryptophan into kynurenines,which drive T-cell differentiation toward an immunosuppressivestate through local depletion of tryptophan (22). Local immunesuppression can therefore act as a mechanism, whereby N-cad-herin escapes an effective immune response. To confirm immune

Figure 2.

IDO1 and N-cadherin expression coincide in clinical prostate cancer. Fluorescent immunostaining in patient samples (n ¼ 8) demonstrates that N-cadherin(CDH2) is associated with IDO1 expression in prostate cancer (A) and high levels of L-Kynurenine (B). A, N-cadherin–negative prostate cancer areas are primarilynegative for IDO1 (top). IDO1 is either expressed by N-cadherin–positive cells (arrowhead) or adjacent tumor cells (arrow) in N-cadherin–positive cancerareas (bottom). B, L-Kynurenine (Kyn) was expressed at medium to high levels in N-cadherin–positive prostate cancer cells in patient samples (n¼ 4). N-cadherin–negative prostate cancer cells generally expressed low levels of L-Kynurenine (asterisk). Hoechst was used to visualize nuclei (blue). Original magnification,�63. Scale bar, 10 mm.

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evasion, we selected IDO1, which was nonlinearly overexpressedin 6 of 8 samples, and investigated its expression and effects on thelocal immunoresponse.

N-cadherin–positive prostate cancer areas show enhancedexpression and activity of IDO1

To validate IDO1 upregulation in N-cadherin–positive tumorregions, we performed fluorescent immunostainings on bothsequenced (n ¼ 5) and independent prostate cancer samples(n ¼ 3; Fig. 2; Supplementary Table S1). In each sample, IDO1was expressed in N-cadherin–positive tumor regions, while ex-pression was rare in tumor areas without N-cadherin (Fig. 2A).IDO1 was either expressed in malignant epithelial cells directlyadjacent to N-cadherin–positive tumor cells, or coexpressed bythe same cancer cells. In addition, we found that IDO1metaboliteL-Kynurenine was elevated in N-cadherin–positive regions, whichindicates enzymatic activity of IDO1 (n ¼ 4; Fig. 2B).

To determinewhether local accumulation of kynurenine is ableto induce N-cadherin expression, we investigated its effects onmRNA expression of N-cadherin (CDH2), E-cadherin (CDH1),and IDO (IDO1) in prostate cancer cell lines (Fig. 3). Stimulationwith 100 mmol/L L-Kynurenine led to 1.4� increase (P¼ 0.05) ofN-cadherin mRNA levels in the N-cadherin–positive cell line PC3(mean � SD; 1.3 � 0.6 vs. 1.8 � 0.3), but did not initiatetranscription in the N-cadherin–negative cell lines DU145 andLNCaP (Fig. 3A). Furthermore, L-Kynurenine stimulation led todownregulated expression of E-cadherin in PC3 (0.9 � 0.3 vs.0.6� 0.1; P¼ 0.05), DU145 (0.3� 0.02 vs. 0.1� 0.01; P¼ 0.03),and LNCaP (2.0 � 0.4 vs. 0.8 � 0.3; P ¼ 0.03; Fig. 3B). Notably,IDO1 expression was decreased upon L-Kynurenine in PC3 cellline (0.6 � 0.2 vs. 0.3 � 0.1; P < 0.01) and suggests a negativefeedback loop, which might finally result in EMT reversal. IDO1was not expressed in DU145 and LNCaP (Fig. 3C).

N-cadherin–positive prostate cancer areas harbor less cytotoxicT cells and more regulatory T cells

Because IDO1 affects differentiation and proliferation ofimmune cells, especially regulatory T cells, we quantified thenumber of CD4þ T helper cells and CD8þ cytotoxic T cells inindependent prostate cancer samples (n ¼ 10; SupplementaryTable S1; ref. 22). Because intraepithelial lymphocytes are criticalto protect the integrity of the epithelial barrier and maintainimmune control, we quantified the number of CD8þ and CD4þ

T cells in the total area of cancer epithelium with adjacent stroma("overall") as well as in cancer epithelium alone ("intraepithe-lial") through IHC double stainings (Fig. 4A and B; refs. 23, 24).We found that the number of intraepithelial CD8þ T cells wassignificantly lower (Fig. 4C; P ¼ 0.04) in N-cadherin–positive(median 2.0; range 1–33) than negative regions (median 4.0;range 1–15). In contrast, the overall number of CD8þ cells washigher (Fig. 4D; P¼ 0.02) in N-cadherin–positive regions (medi-an 11.5; range 1–53) than negative regions (median 7.9; range1–37). The number of intraepithelial CD4þ T cells was notsignificantly different between N-cadherin–positive versus nega-tive regions (Fig. 4E; P ¼ 0.21). The overall number of CD4þ Tcells, however, was slightly higher in N-cadherin–positive tumorregions (median 13; range 3–79) as compared with N-cadherinnegative tumor areas (median 11; range 3–70; Fig. 4F; P ¼ 0.02).Finally, immunofluorescent triple stainings with Hoechst, CD4,FOXP3, and CDH2 (Fig. 5A) indicated that the increase of CD4þ

T cells coincided with the presence of a CD4þ/FOXP3þ T regu-latory phenotype in N-cadherin–positive prostate cancer regions(Fig. 5B), although the sample size was too small for statisticalanalysis (n¼ 3). Together, these results indicate that expression ofN-cadherin and IDO1 leads to an increased number of CD4þ

(regulatory) T cells with a concordant decrease of intraepithelialCD8þ cytotoxic T cells, and indicates immune evasion.

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Figure 3.

L-Kynurenine induced N-cadherinupregulation and E-cadherindownregulation in prostate cancer celllines. Stimulation (n ¼ 4) of threeprostate cancer cell lines with 100mmol/L L-Kynurenine (Kyn) inducedEMT marked by significantupregulation of N-cadherin (CDH2) inPC3 (A) and downregulation ofE-cadherin (CDH1) expression in PC3,DU145, and LNCaP (B). IDO1 wasdownregulated upon L-Kynureninestimulation (C). Gene expression wascorrected for the expression ofhousekeeping genes (HKG). Mean andSD are shown.






DiscussionEMT is a biological process involved in tumor invasion,

resistance to chemotherapy, and metastasis (25–27). WhileEMT has extensively been investigated in vitro, it is unclear

what mechanisms drive EMT in patients with cancer. In thisstudy, we have shown that EMT, characterized by N-cadherinexpression in ill-formed tumor glands, is related to localenrichment of immunosuppressive molecules such as IDO1

Figure 4.

N-cadherin–positive tumor areasharbor less intraepithelial cytotoxicCD8þ T cells. The number of CD8þ andCD4þ T cells was scored inN-cadherin–negative (Ncad�) andN-cadherin–positive (Ncadþ) areasthrough IHC double staining. Redstaining from alkaline phosphataseindicates CD8 (A)- or CD4 (B)-positive cells (arrows), whereas brownstaining from DAB indicatesexpression of N-cadherin. Eight areaswith a total size of 0.5 mm2 werescored per condition and per patient(n¼ 10) for CD8þ (C and D) and CD4þ(E and F) cells in prostatic cancerepithelium (C and E) or overall(epithelium and adjacent stroma; Dand F). Quantification of CD8þ andCD4þ cells demonstrated that thenumber of intraepithelial cytotoxicCD8þ T cells (C), but not CD4þ cells(E), was decreased in N-cadherin–positive cancer epithelium (Ncadþ).The overall number of both CD8þ (D)and CD4þ (F) cells was higher inN-cadherin–positive areas.

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and immune cells such as regulatory T cells and cytotoxic T cellsin clinical prostate cancer. At a mechanistic level, we demon-strated enhanced enzymatic activity of IDO1 in tissues led todownregulated expression of E-cadherin and upregulatedexpression of N-cadherin in prostate cancer cell lines. Theconcomitant enrichment of CD4þ/FOXP3þ regulatory T cellsand reduction of intraepithelial CD8þ cytotoxic T cells, indi-cates an intimate interaction between EMT as marked byN-cadherin expression and local evasion of an effective anti-tumor CD8 T-cell response in prostate cancer.

Although the exact mechanisms of EMT and immune cellresponse in patients with cancer are still largely unknown, recentstudies indicate an interaction between both essential biologicalprocesses (28, 29). Breast cancer cells that undergo EMT areassociated with an immunosuppressive phenotype and weremore resistant to immunotherapy than epithelial tumors (28).Inflammatory cytokine-induced EMT significantly upregulatedIDO1 in lung, breast, and liver cancer cell lines (30). Chen andcolleagues demonstrated that IDO-induced kynurenine expres-sion led to activation of the aryl hydrocarbon receptor (AhR)complex, resulting in E-cadherin degradation in breast cancer(31). In hepatocellular carcinoma cell lines, EMT-associated N-cadherin upregulated expression was abolished through silencingof the AhR (32). Although the expression of AhR mRNA was not

significantly affected in our study, this does not exclude an alteredexpression or activation of the protein.

Our findings are in line with previous studies and provideevidence that interaction between EMT and an immunosup-pressive response exists in patients with clinical prostate cancer(28–32). While IDO1 activation was present in N-cadherinpositive tumor areas and associated with a suppressive immuneprofile, the primary EMT-initiating step in prostate cancer is notyet clear. Pathway analysis revealed TGFb and IFNg signaling asputative upstream regulatory pathways (see SupplementaryTable S5). TGFb can be secreted by both stromal and immunecells, and is able to induce both EMT and IDO1 expressionthrough activation of both Smad-dependent and -independentpathways (33, 34). Binding of TGFb to its receptor activates thenoncanonical nuclear factor-kB pathway and generates a pos-itive feedback loop that stimulates IDO1 expression in a phos-phatidylinositol-3-OH kinase (PI3K) and Src homology region2 domain-containing phosphatase-1 (SHP-1)–dependent man-ner. In our dataset TGFB1, PTPN6 (SHP-1), and IDO1 were allsignificantly upregulated in N-cadherin–positive regions. Incontrast, we did not find differential expression of Smad genes.This suggests that Smad-independent TGFb signaling plays arole in the accompanied local induction of both EMT andimmunosuppression in clinical prostate cancer.

Figure 5.

The number of T regulatory cells(CD4þ/FOXP3þ) is increased in N-cadherin–positive prostate cancer.Fluorescent immunostaining wasperformed to visualize and quantify thenumber of CD4þ/FoxP3þ regulatory Tcells in N-cadherin (CDH2)–negativeand –positive regions in patientsamples (n ¼ 3). A, DAPI staining wasused to identify nuclei (blue) combinedwith fluorescently labeled CD4 (green),CDH2 (red), and FoxP3 (magenta). Thecomposite image demonstrates aCD4þ and FOXP3þ regulatory T cell(arrow) in close proximity to anN-cadherin–positive prostate cancercell.B, The number of T regulatory cellswas scored. Matching symbolsindicate paired samples. The totalnumber of regulatory T cells wasincreased in N-cadherin–positive(Ncadþ) tumor regions compared withN-cadherin–negative regions (Ncad-)in all three patient samples. Originalmagnification, �63.






The inflammatory cytokine IFNg has been reported to initiatethe start of negative feedback loops that result in enhancedexpression of regulators of immune responses, such as IDO1,PD-L1, and regulatory T cells in melanoma (35). Alongside thisprocess, cancer cells may demonstrate enhanced activity of Wntand PI3K pathways, which in their turn, advocate further loss ofimmune control and generally results in loss of numbers andactivity of CD8 effector T cells within tumor tissue (36, 37). Thereduced number of CD8 effector T cells within cancer epitheliumcombined with the overall (epithelium and stroma combined)increased number of CD8 effector T cells, suggests an immuneevasion mechanism deployed by tumors (5). This acknowledgedimmune evasion mechanism describes the exclusion of CD8þ Tcells from the tumor and thereby allows cancer cells to avoidimmune destruction.

In this study, there was no significant change in expression oftranscription factors associated with EMT in vitro such as SNAIL,SLUG, TWIST, ZEB1, and ZEB2 (16–21). A putative explanationis that these transcription factors have mostly been studied in cellcultures models based on metastasized and androgen-indepen-dent cells lines that represent end-stage prostate cancer. However,in this study we investigated clinical hormone-na€�ve prostateadenocarcinoma that infiltrates and interacts with preexistentprostate stromal tissue. Taken together, this paradoxical findingcould indicate that alternativemechanisms are involved in EMT inearly- and late-stage disease.

The strength of this study is the primary analysis of EMT inclinical prostate cancer specimens. Microdissection of morpho-logically ill-defined areas that expressed N-cadherin allowed adetailed study of biological events in specific tumor areas. Whileuse of whole tissue slides for molecular studies is less laborious,specific local molecular biological events might be diluted andremain undiscovered. On the other hand, application of stringentmorphologic and IHC criteria for LCM selection combined withthe relatively rare occurrence of EMT, hampered analysis on a largenumber of tissue samples. In addition, we used N-cadherinexpression as IHC marker for EMT, which does not cover the fullrange of EMT in prostate cancer, namely those EMT-like processesthat downregulate epithelial markers without N-cadherin expres-

sion. Finally, the role of other enzymes involved in tryptophandegradation, such as IDO2 and TDO2, remains to be elucidated.

In conclusion, we found that N-cadherin expression in clinicalprostate cancer is enriched for enzymatic activity of IDO1 result-ing in local accumulation kynurenine. L-Kynurenine may induceor sustain an immunosuppressive microenvironment that ischaracterized by increased regulatory FOXP3þ/CD4þ T cells anddecreased intraepithelial cytotoxic CD8þ immune cells. Togetherthesefindings reveal an important link betweenEMTand immuneevasion in patient with prostate cancer.

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

Authors' ContributionsConception and design: K. Kolijn, G.J.L.H. van LeendersDevelopment of methodology: K. Kolijn, G.W. Jenster, G.J.L.H. van LeendersAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Kolijn, E.I. Verhoef, G.J.L.H. van LeendersAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis): K. Kolijn, M. Smid, R. B€ottcher, R. Debets,G.J.L.H. van LeendersWriting, review, and/or revision of the manuscript: K. Kolijn, E.I. Verhoef,M. Smid, R. B€ottcher, G.W. Jenster, R. Debets, G.J.L.H. van LeendersAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Kolijn, E.I. VerhoefStudy supervision: G.J.L.H. van Leenders

AcknowledgmentsThis research was sponsored by the Dutch Cancer Society (EMCR2011-5006

to K. Kolijn and E.I. Verhoef).We thank A.M. Hoogland for instructions on laser capture microdissection

and the ErasmusMCTissue Bank for storage and supply of frozen prostate tissuesamples. We kindly thank J.A. Stoop for his support on dual chromogenic IHC.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 4, 2017; revised April 16, 2018; accepted June 13, 2018;published first June 19, 2018.

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2018;78:4671-4679. Published OnlineFirst June 19, 2018.Cancer Res Kimberley Kolijn, Esther I. Verhoef, Marcel Smid, et al. ExpressionDemonstrates Enhanced Immune Evasion Marked by IDO1

Mesenchymal Transition in Human Prostate Cancer−Epithelial

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Epithelial Mesenchymal Transition in Human Prostate Cancer ...Cancer Res; 78(16); 4671–9. 2018 AACR. Introduction Prostate cancer is a heterogeneous disease in terms of patho-logic