Notch-Induced Myeloid Reprogramming in Spontaneous Pancreatic Ductal Adenocarcinoma by Dual Genetic Targeting Phyllis F

Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0052

Cancer Tumor Biology and Immunology Research

Notch-Induced Myeloid Reprogramming in Spontaneous Pancreatic Ductal Adenocarcinoma by Dual Genetic Targeting Phyllis F. Cheung1,2, Florian Neff1,2, Christian Neander1,2, Anna Bazarna1,2, Konstantinos Savvatakis1,2, Sven-Thorsten Liffers1,2, Kristina Althoff1,2, Chang-Lung Lee3, Everett J. Moding3, David G Kirsch3, Dieter Saur2,4, Alexandr V. Bazhin5,6, Marija Trajkovic-Arsic1,2, Mathias F. Heikenwalder7, and Jens T. Siveke1,2,4

Abstract

Despite advances in our understanding of the genetics of Notch2-IC or deletion of Rbpj. Myeloid-specific Notch acti- pancreatic ductal adenocarcinoma (PDAC), the efficacy of vation significantly decreased tumor infiltration by protumori- therapeutic regimens targeting aberrant signaling pathways genic M2 macrophages in spontaneous endogenous PDAC, remains highly limited. Therapeutic strategies are greatly ham- which translated into significant survival benefit. Further char- pered by the extensive desmoplasia that comprises heteroge- acterization revealed upregulated antigen presentation and neous cell populations. Notch signaling is a contentious cytotoxic T effector phenotype upon Notch-induced M2 reduc- pathway exerting opposite roles in tumorigenesis depending tion. This approach is the first proof of concept for genetic on cellular context. Advanced model systems are needed to targeting and reprogramming of myeloid cells in a complex gain more insights into complex signaling in the multilayered disease model of PDAC and provides evidence for a regulatory tumor microenvironment. In this study, we employed a dual role of Notch signaling in intratumoral immune phenotypes. recombinase-based in vivo strategy to modulate Notch signaling specifically in myeloid cells to dissect the tumorigenic role Significance: This study provides insight into the role of of Notch in PDAC stroma. Pancreas-specific KrasG12D acti- myeloid-dependent NOTCH signaling in PDAC and accent- vation and loss of Tp53 was induced using a Pdx1-Flp trans- uates the need to dissect differential roles of signaling path- gene, whereas Notch signaling was genetically targeted using a ways in different cellular components within the tumor micro- myeloid-targeting Lyz2-Cre strain for either activation of environment. Cancer Res; 78(17); 4997–5010. 2018 AACR.

Introduction cells, and extracellular matrix (1, 2). Recent studies have revealed crucial role of stromal components in promoting the induction Pancreatic ductal adenocarcinoma (PDAC) is one of the most and progression of tumorigenesis (1, 3–5). Comprehensive stud- aggressive malignancies with no effective therapeutic options for ies on differential roles of signaling pathways in regulating tumor long-term tumor control thus far. Therapeutic targeting of PDAC cells and stromal components in PDAC will advance our under- focusing on deregulated molecular signaling pathways in cancer standing of the complicated pathogenesis. cells has been largely disappointing. One of the hallmarks of Notch signaling is a key developmental pathway that is known PDAC is the presence of extensive desmoplasia, which comprises to regulate cell proliferation, apoptosis, as well as tumorigenesis heterogeneous cell populations including fibroblasts, immune of various solid tumors including pancreatic cancers (6–8). How- ever, the role of Notch signaling in PDAC still remains highly 1Division of Solid Tumor Translational Oncology, West German Cancer Center, contentious. Notch receptors and ligands, and their downstream University Hospital Essen, Essen, Germany. 2German Cancer Consortium (DKTK, targets have been frequently reported to be overexpressed in partner site Essen) and German Cancer Research Center, DKFZ, Heidelberg, PDAC, suggesting a role in PDAC development and progression Germany. 3Department of Radiation Oncology, Duke University Medical Center, (7, 9). Activation of Notch1-IC led to the progression of 4 Durham, North Carolina. Medical Department, Klinikum rechts der Isar, Tech- preneoplastic lesions in a genetic mouse model (10), while € € 5 nische Universitat Munchen, Munich, Germany. Department of General, Visceral another study showed that genetic ablation of Notch1 in a mouse and Transplant Surgery, Ludwig-Maximilians University, Munich, Germany. 6German Caner Consortium (DKTK), Partner Site Munich, Germany. 7Division model of KRAS-induced PDAC resulted in an increase in high- of Chronic Inflammation and Cancer, DKFZ, Heidelberg, Germany. grade pancreatic intraepithelial neoplasia (PanIN) lesions (11). We described an oncogenic role of Notch2 but not Notch1 in Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). regulating PanIN progression and tumor differentiation (12). Notably, phase II clinical trials assessing the use of blocking P.F. Cheung and F. Neff are co-first authors of this article. antibodies against Notch2/3 or the Notch ligand Dll4 in combi- Corresponding Author: Jens T. Siveke, West German Cancer Center and DKTK nation with chemotherapy have reported no benefit or even partner site Essen, University Hospital Essen, Hufeland, Essen 45147, Germany. detrimental outcomes (13, 14), therefore casting doubts onto Phone: 4920-1723-3704; Fax: 4920-1723-6725; E-mail: [email protected] the efficacy of Notch as a therapeutic target in PDAC. doi: 10.1158/0008-5472.CAN-18-0052 Given the importance of stromal components in tumorigene- 2018 American Association for Cancer Research. sis, Notch might exert either oncogenic or tumor-suppressive role

www.aacrjournals.org 4997

Cheung et al.

depending on cellular context. Indeed, impairment of Notch Isolation of bone marrow–derived cells and macrophage signaling in epidermis and fibroblasts was shown to result in differentiation profound changes in stroma, leading to induction of tumor Bone marrow–derived cells were isolated from femur and tibia development (15, 16). Increasing evidence has indicated that the of mice aged between 6 and 12 weeks. Bone marrow was collected Notch-mediated tumor suppression might be at least partially by flushing with RPMI medium (Life Technologies). Red blood mediated by regulating inflammation in the tumor microenvi- cells (RBC) were lysed with RBC Lysing Buffer (Sigma-Aldrich ronment (15, 17). Ablation of Notch in mice significantly upre- GmbH) for 5 minutes at room temperature, washed, and then gulated proinflammatory cytokine expression (18, 19). Intrigu- resuspended in macrophage medium (RPMI, 15% FBS, 5% horse ingly, deletion or inactivation of Notch signaling induced inflam- serum, 1% NEAA, 1% sodium pyruvate; Life Technologies) con- matory responses, which suppressed tumorigenesis through the taining 50 ng/mL murine M-CSF (PELOBiotech) for 7 days. The antitumor function of T cells present in the inflammatory milieu uncoated surface of petri dishes allows attachment of highly (20, 21). Collectively, these studies demonstrated that Notch adherent cells as macrophages, while floating cells were removed signaling in stromal cells could induce tumor-suppressive effects by rinsing with PBS. Macrophage phenotype and purity were through mediating inflammation in the tumor microenviron- assessed based on CD11b and F4/80 expression using flow ment. However, precise cellular and molecular mechanisms cytometry. underlying these phenomena still need further examination. Tumor-associated macrophages (TAM) represent the most Induction of Cre-dependent LoxP site recombination and abundant immune cell population in the tumor microenviron- polarization in vitro ment, and dysregulated polarization facilitates tumor growth and Bone marrow–derived macrophages (BMDM) were treated metastasis of PDAC (22–24). In patients with PDAC, increased with 1 mmol/L recombinant NLS-His-Tat-NLS-CRE protein (36) tumor-promoting M2 TAMs are associated with significantly in serum-free RPMI medium 1:1 diluted in PBS overnight. Culture shorter patient survival (25–28). Recently, Notch signaling was supernatant was removed the next day and then changed to shown to regulate myeloid cell differentiation (29), potentially complete macrophage medium. To induce M1 polarization, cells via transcriptional repression of M2-associated genes by Notch were treated with 1 mg/mL LPS (Sigma-Aldrich) for 6 or 24 hours, activation (30), and upregulation of M2-associated genes via whereas for M2, cells were treated with 10 ng/mL recombinant posttranslational modification of IRAK2 (31). However, the rel- murine IL4 (eBioscience) for 72 hours. evance of these findings in complex disease models of cancer such as in PDAC has not been addressed so far. Moreover, the role of Isolation of tumor cells from endogenous PDAC Notch signaling in myeloid cells in tumor formation and pro- Tumors were minced and then digested in 1 mg/mL collagenase gression has not been investigated. type V (Sigma-Aldrich GmbH) for 45 minutes at 37C. The cells To study the context and spectrum of immune–tumor inter- were filtered through a 100-mm cell strainer. RBCs were lysed by actions, endogenous immunocompetent tumor models are RBC lysing buffer for 5 minutes at room temperature. Cells were necessary. Genetically engineered mouse models based on then washed, filtered through 40-mm cell strainer, and subject to pancreas-specific activation of oncogenic KRAS resemble subsequent experiments. human PDAC in many aspects including formation of an extensive fibro-inflammatory stromal reaction (32). To target Flow cytometric analysis and cell sorting nonpancreatic and nontumoral cell lineages, Schonhuber and Multicolor flow cytometry experiments were performed using colleagues introduced a dual-recombinase approach using a Beckman Coulter Gallios flow cytometer (Beckman Coulter), Pdx1-Flp transgene to target KRAs and Tp53 (33), allowing while cell sorting was performed using BD FACSAria III (BD selective Cre-based targeting of the tumor microenvironment. Biosciences). All samples were incubated with CD16/32 antibody In this study, we investigated the role of Notch signaling in (BD Biosciences) to block unspecific FC receptor–mediated anti- TAM polarization in PDAC development and progression using body binding prior to antibody incubation. Dead cells were a dual-recombinase gene targeting strategy as a genetic proof- excluded by staining with Fixable Viability Dye 780 (eBioscience), of-concept approach. LIVE/DEAD Fixable Yellow stain (Life Technologies), or propi- dium iodide (PI) (Clontech). For intracellular staining, cells were fixed with 2% PFA/PBS after extracellular staining. Cells were then Materials and Methods permeabilized with 0.5% Saponin/PBS prior to antibody incu- Mouse strains and tumor models bation. For FOXP3 staining, the Foxp3/Transcription Factor Buffer fl fl Unless otherwise stated, Ptf1awt/Cre;Kraswt/LSL-G12D;p53 / (CKP) Set (eBioscience) was applied. After washing, cells were subjected was used as tumor model of spontaneous PDAC for phenotypic to flow cytometric analysis. The antibody list is shown in characterization and quantification of immune cells. For the dual- Supplementary Table S2. Raw data were analyzed using FlowJo recombinase system, a myeloid-specific Cre-line (Lyz2wt/Cre) was software version 7.5.5 (Tree Star Inc.). fl fl crossed to R26wt/LSL-N2IC and Rbpj / mice (34, 35). Lyz2wt/Cre; fl fl R26wt/LSL-N2IC (Lyz2;N2IC) and Lyz2wt/Cre;Rbpj / (Lyz2;Rbpj) IHC lines were crossed to the Pdx1-Flp;Kraswt/FSF-G12D;p53frt/frt (FKP) IHC staining was performed using the Dako REAL Alkaline or Pdx1-Flp;Kraswt/FSF-G12D;p53wt/frt (FKPhet) model. Details of Phosphatase or Peroxidase Detection System (Dako), following original and interbred mouse strains are listed in Supplementary the manufacturer's instructions. Antigen retrieval on formalin- Table S1A and S1B. All animal protocols were performed accord- fixed paraffin-embedded (FFPE) sections was performed by heat- ing with appropriate guidelines and the experimental protocols induced epitope retrieval using citrate buffer (pH6) for CD11b, were approved by the local Animal Use and Care Committee at the hCD2, MRC1, NK1.1, collagen, MHCII, CD11c and CD80; Klinikum Rechts der Isar of the TU Munchen,€ Germany. Tris/EDTA (pH9) for CD3, CD4, CD8, Eomes, T-bet and Lag3,

4998 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

and proteinase K treatment for F4/80. After blocking with serum- mented in each experiment to prove single product amplification. free protein blocking solution (Dako), slides were incubated for Data were analyzed using DCt calculations where RPLP0 or primary antibodies for 1 hour at room temperature, secondary cyclophilin A served as housekeeper control for normalization. antibody for 30 minutes at room temperature, and then subjected The amplification efficiency was experimentally determined to Fast Red or DAB chromogen development. The antibody list is or assumed as 2 (doubling each cycle). Relative mRNA shown in Supplementary Table S3. Slides were then counter- expression levels compared with housekeeper gene expression stained with hematoxylin, dehydrated, and mounted. Stromal (efficiency-DCt) were used for visualization. content and acinar cells were determined by Movat's pentachrome staining following the manufacturer's protocol (modified accord- NanoString nCounter RNA expression analysis ing to Verhoeff, Morphisto GmbH). The PanCancer Immune Profiling Panel (NanoString Technol- Slides were scanned and digitalized by Zeiss Axio Scanner Z.1 ogies Inc.), which includes 730 immune-related genes and 40 (Carl Zeiss AG) with 5 and 10 objective magnification. The housekeeping genes, was used in the study. Expression data were percentage of positive cells for IHC staining, while the area of normalized and analyzed with the nSolver Advanced Analysis collagen, stroma (ground substance/mucin stained bluish, colla- Software 1.1.4 using the PanCancer Immune Profiling Advanced gen stained yellowish) and/or acinar cells (deep red) for Movat's Analysis Module (NanoString Technologies). For background pentachrome staining were quantified by Definiens (Definiens correction, the mean count of negative controls plus two times AG) as the average of 5 representative fields (10 objective the SD was subtracted from the counts for each gene. The geNorm magnification) captured from each tissue section of each mouse. algorithm was used to identify the most stable housekeeping For quantification, total number of cells in each field was deter- genes. The geometric mean of the selected housekeeping genes mined based on nuclei staining (hematoxylin for IHC; DAPI for was used to calculate a normalization factor for each sample. The immunofluorescence staining) detected by the software. The cell-type–specific scores were calculated as mean log2 value of average number of positive cells (percentage in total number of characteristic genes for corresponding immune cell types. When- cells in each field) from the 5 representative fields was taken as the ever stated, relative score was presented as the relative abundance percentage of positive cells for each individual mouse. For quan- of certain immune subset with regard to either total tumor- tification of Ki67 and cleaved caspase-3, the 5 representative fields infiltrating leukocytes (TIL) or total T cells. The pathway scores were captured in the area of tumor cells as we aimed to show the were analyzed by the first principal component (PC). For a given proliferation and apoptosis of tumor cells upon myeloid-specific pathway, PC analysis scored each sample using a weighted average Notch modulation. Tumor cells could be distinguished based on of its gene expression values. their sizes and histology (infiltrating immune cells are largely found in stroma). Besides, area of extensive immune cell infiltra- Statistical analysis tion, for example, infiltrating lymph nodes or tertiary lymphoid All statistics were calculated using GraphPad Prism 6.0 (Graph- structures, was avoided. Pad Software). Two-tailed nonparametric Mann–Whitney test was applied for all analysis, except for survival data by log-rank Immunofluorescence staining (Mantel–Cox) test, and correlation analysis by Spearman rank FFPE sections were deparaffinized and then fixed with form- correlation coefficient. aldehyde:methanol (1:10) prior to antigen retrieval by heat- induced epitope retrieval using citrate buffer (pH6) or Tris/EDTA (pH9). Each section was put through several sequential rounds of Results staining; each includes a protein blocking followed by primary M2-phenotype TAM is the predominant immune subset in antibody and corresponding secondary horseradish peroxidase– PDAC microenvironment conjugated polymer (Zytomed Systems or PerkinElmer). Each We first studied the dynamics of tumor–stroma interactions at horseradish peroxidase–conjugated polymer mediated the cova- different time points during tumor progression in Ptf1awt/Cre; fl fl lent binding of a different fluorophore using tyramide signal Kraswt/LSL-G12D;p53 / mice (named CKP hereafter; for details, refer amplification. The sequential multiplexed staining protocol is to Supplementary Table S1), which show a highly aggressive shown in Supplementary Table S4. Such covalent reaction was clinical course and display prominent desmoplasia (37). Pancre- followed by additional antigen retrieval in heated citric buffer atic tissue was harvested at different stages: preneoplasia (pH6) or Tris/EDTA (pH9) for 20 minutes to remove antibodies (<4 weeks of age), early (4–6 weeks), and advanced PDAC before the next round of staining. After all sequential staining (10 weeks). IHC staining showed that the number of proliferative þ reactions, sections were counterstained with DAPI (Vector lab). Ki67 tumor cells significantly increased from preneoplasia to Slides were scanned and digitalized by Zeiss Axio Scanner Z.1 early PDAC, and then decreased slightly at advanced stage (Carl Zeiss AG) with 10 objective magnification. (Supplementary Fig. S1A). A similar trend was observed for apoptotic marker cleaved caspase-3, although the frequency was Real-time quantitative RT-PCR relatively low (Supplementary Fig. S1A). Tumor histology as qPCR was performed by Roche LightCycler 480 using Light- revealed by Movat's pentachrome stain demonstrated that acinar Cycler 480 SYBR Green I Master Kit (Roche GmbH). Primers for cells significantly decreased, whereas desmoplasia increased from qRT-PCRs were designed using the NCBI Primer Blast and pur- preneoplasia to advanced stage PDAC (Fig. 1A). chased from Eurofins MWG Operon GmbH. The primer list is Tumor-infiltrating immune cells are a major component of shown in Supplementary Table S5. The PCR products were desmoplasia. We profiled the dynamics of immune subsets along þ designed with a size of approximately 100 bp. All real-time qPCR PDAC progression. A significant percentage of CD45 leukocytes experiments were run under 58C annealing condition and (20%) was detected in preneoplasia, and increased in early amplification was run for 45 cycles. A melting curve was imple- PDAC (40%), but then dropped to approximately 30% at

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 4999

Cheung et al.

Figure 1. M2 macrophages predominated tumor-infiltrating leukocytes in CKP tumors. A, Representative Movat, CD45, CD19, CD3, CD11b, F4/80, and MRC1 staining of different tumor stages: preneoplasia (<4 weeks), early (4–6 weeks), and advanced (>8 weeks) PDAC in CKP mice (n 5/group). Magnification, 25; 200. Scale bar, 100 mm. Right, percentage of positive cells or area (for Movat's pentachrome staining only) as the average of 5 fields from each mouse (objective magnification, 10). Mean þ SD is shown. B, Flow cytometric analysis on the immune phenotype of spleens from wild-type or end-stage CKP mice. Percentage of viable CD45þ cells was pregated for T cells (CD3þ), B cells (CD19þ), and myeloid cells (CD11bþ) quantification. Percentage of CD4þ and CD8þ cells in total CD3þ T cells. Na€ve, hi hi hi hi þ þ CD44loCD62L ; EM (effector memory), CD44 CD62Llo; CM (central memory), CD44 CD62L in total CD4 and CD8 T cells were quantified. WT, n ¼ 6; CKP, n ¼ 6. C, PDAC tumors of end-stage CKP mice were digested into desegregated cells and analyzed by flow cytometry. Live CD45þ cells were gated as viable leukocytes for subsequent immune subpopulation characterization: T cells (CD3þ), B cells (CD19þ), and myeloid cells (CD11bþ); CD4þ T (CD3þCD4þCD8), CD8þ T þ þ þ þ þ þ þ þ þ þ þ þ þ (CD3 CD4 CD8 ), Treg (CD3 CD4 Foxp3 CD25 ), gdT (CD3 gdTCR ), B1 (CD19 CD43 ) and B2 (CD19 CD43 ), NK (NK1,1 ), M-MDSCs (CD11b F4/ hi þ hi þ þ þ þ þ 80 /loGR1 /loLy6C ), G-MDSCs (CD11b F4/80 GR1 ), and TAM (CD11b F4/80 GR1 /lo). TAMs were further dissected into M1 (iNOS ) and M2 (MRC1 , ARG1 ). n ¼ 12, except for Treg and NK (n ¼ 5), gdT(n ¼ 3), M-MDSC (n ¼ 7). Mean þ SD are shown. , P < 0.05; , P < 0.005.

5000 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

þ advanced stage (Fig. 1A). CD3 T cells demonstrated a similar duction of effector cytokines such as IFN-suppressed. Eomes þ þ trend as CD45 cells, whereas CD19 B cells were almost unde- regulates cytolytic gene program in CTLs, and T-bethiEomeshi cells tectable in preneoplastic lesions, but then continuously increased represent fully differentiated CTL populations (39). IHC staining along PDAC progression (Fig. 1A). However, frequencies of both showed that both transcription factors and LAG3 were found þ þ CD3 T and CD19 B cells were relatively low (less than 5% and at low expression levels in the tumor-infiltrating T cells (Supple- 1.5%, respectively). On the contrary, a substantial number of mentary Fig. S1A), and absent in normal pancreas (Supplemen- þ CD11b myeloid cells was found in preneoplasia (15%), and tary Fig. S1B). increased to approximately 20% in early and advanced PDAC, Because T-bet and Eomes are also implicated in the cytotoxic respectively (Fig. 1A). Further characterization showed that functions of NK cells, we assessed NK infiltration in PDAC þ F4/80 macrophages comprised the major subpopulation of development. IHC analysis showed that NK cells were absent in þ þ CD11b myeloid cells (Fig. 1A). Among F4/80 macrophages, preneoplastic lesions. However, NK cells were detected in early þ MRC1 M2 macrophages represented the dominant subpopula- and late PDAC, albeit at low frequency (<1%; Supplementary þ tion (Fig. 1A), whereas iNOS M1 macrophages were rarely Fig. S1A). On the basis of the expression patterns of T-bet, Eomes, observed in all stages (<1.5%, Supplementary Fig. S1A). In normal and NK1.1 in consecutive tissue sections, NK cells might also pancreas, the above markers are all almost absent (Supplementary contribute to the expression of T-bet and Eomes in early and late Fig. S1B). PDAC (Supplementary Fig. S1A). However, because NK-cell þ Flow cytometric analysis was performed to quantify various frequency was relatively low when compared with CD3 T cells immune subpopulations in advanced CKP mice. First, systemic (Fig. 1A), the increased T-bet and Eomes expression should be immunoregulatory effects during PDAC progression were largely contributed by T cells. þ þ þ assessed by measuring CD3 T cells, CD19 B cells, and CD11b myeloid cells in the spleens of wild-type and CKP mice. Spleens of Notch activation counteracts IL4-induced M2 polarization in CKP mice showed a significant increase of myeloid cells with a vitro reduction of B cells compared with those of wild-type mice Predominant M2 TAMs might be associated with defective (Fig. 1B). However, the number of total and different subsets T-cell responses in PDAC. Therefore, we investigated regulatory of T cells was not affected by the presence of pancreatic mechanisms for M2 polarization. The Notch signaling pathway neoplasms (Fig. 1B). has been reported to regulate macrophage polarization, in Next, we characterized the immune landscape in advanced CKP which Notch activation led to M2-associated gene repression þ tumors. Approximately 60% of CD45 leukocytes were positive (30, 31). þ for the myeloid lineage marker CD11b, whereas CD3 T cells and First, we studied the effects of manipulating the Notch pathway þ CD19 B cells accounted for 15% and 20% of leukocytes, respec- using BMDMs. Bone marrow–derived cells were treated with tively (Fig. 1C). Further analysis was performed to comprehen- M-CSF for 8 days, and then confirmed for macrophage phenotype sively delineate the composition of tumor-infiltrating immune by expression of both CD11b and F4/80 (Supplementary Fig. S2). cells (detailed gating strategies as shown in Supplementary To test the ability of BMDMs to undergo CRE-mediated recom- þ þ þ Fig. S1C–S1G). CD4 and CD8 T cells contributed to CD3 bination, BMDMs from R26LSL-tdTomato reporter mice were treated population equally (7% of total TILs). Regulatory T cells (Treg), with recombinant NLS-TAT-CRE (40). Approximately, 95% of gd T cells, and natural killer (NK) cells accounted for only 1% cells successfully underwent genetic loxP-site recombination as each (Fig. 1C). Conventional CD43 B2 cells represented determined by tdTomato fluorescence detected by flow cytometry þ þ 15% of leukocytes, but no CD19 CD5 CD1dhi Bregs were and fluorescence microscopy (Fig. 2A). found in tumors or spleens. TAMs, characterized by a To study the effect of Notch activation in M2 polarization, þ þ þ LSL-N2IC CD45 CD11b GR1 /loF4/80 phenotype, were the largest BMDMs from R26 mice (34) were pretreated with or myeloid subpopulation (Fig. 1C). Myeloid-derived suppressor without recombinant CRE protein, and subsequently stimulated þ cells (MDSC) accounted for 10% of all CD45 cells, with with IL4 to induce M2 polarization. In this model, CRE recombi- dominance of granulocytic (G-MDSC) over monocytic MDSCs nase excises a transcriptional STOP-cassette, resulting in consti- (M-MDSC). Further characterization on TAMs revealed a clear tutive expression of transcriptionally active Notch2-IC and þ þ enrichment of MRC1 and ARG1 M2 phenotypes (Fig. 1C). human CD2, a coexpressed reporter molecule (Fig. 2B). CRE- dependent activation of Notch signaling was confirmed by hCD2, Intratumoral T cells do not express markers for effector as detected by flow cytometry (Fig. 2C). IL4 strongly induced the functions expression of M2-associated genes Mrc1, Mgl1, and Arg1, whereas Although tumor antigens can elicit T cell–mediated antitumor no significant change was observed for M1-associated genes (Fig. response, T cells are frequently prevented from eradicating tumor 2D). The IL4-induced upregulation of Mrc1, Mgl1, and Arg1 cells by various immunosuppressive programs (24, 38). IHC expression was significantly abrogated by concomitant Notch þ þ staining showed that CD4 and CD8 cells were rarely observed activation (Fig. 2D). In addition, effect of Notch activation on in preneoplastic lesions. Although they increased as tumor pro- MRC1 protein level was validated by flow cytometry (Fig. 2E), gressed, the frequency remained low (less than 1% and 2%, supporting the observation that Notch activation counteracts IL4- respectively; Supplementary Fig. S1A). Given the M2 predomi- induced M2 polarization. nated PDAC microenvironment, we hypothesized that the anti- To explore the effect of Notch activation in M1 polarization, tumor activity of infiltrating T cells might be suppressed. We thus BMDMs were stimulated with LPS following treatment with assessed the expression of two critical transcription factors for recombinant CRE protein. LPS alone induced an upregulation effector functions of T cells, T-bet, and Eomes, as well as the T-cell of iNos, IL1b, IL6, IL12a, and IL12b genes. However, combination activation marker lymphocyte activation gene-3 (LAG3). T-bet is of LPS and CRE did not exert additional effect on M1 gene induced upon na€ve T-cell priming and is required for the pro- upregulation or M2 gene reduction (Fig. 2F). The result was

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 5001

Cheung et al.

Figure 2. Notch modulation regulates macrophage polarization in vitro. A, BMDMs isolated from R26LSL-tdTomato reporter mice treated with 1 mmol/L recombinant NLS-TAT-CRE overnight and analyzed for tdTomato fluorescence on day 4 by flow cytometry or observed under microscope. Scale bar, 100 mm. B, Simplified description of genetic R26LSL-N2IC construct. C, In vitro LoxP recombination assessed by detecting hCD2 on N2IC BMDMs. D and E, BMDMs from R26LSL-N2IC stimulated with 10 ng/mL IL4 for 72 hours with or without previous CRE treatment. D, Relative expression levels of M1 and M2 marker genes determined by qRT-PCR (n ¼ 6). Mean þ SD is shown. E, Representative flow cytometric analysis showing MRC1 level on CD11bþF4/80þ cells upon CRE and IL4 treatment. F and G, BMDMs LSL-N2IC from R26 stimulated with 1 mg/mL LPS for 6 hours (F) or overnight (G) with or without previous CRE treatment. F, Relative mRNA expression levels of M1 and M2 marker genes determined by qRT-PCR. n ¼ 6, except for þCRE-LPS: n ¼ 3. G, Flow cytometry analysis showing percentage of iNOSþ and MRC1þ cells in F4/80þ BMDMs. n ¼ 4. Mean þ SD is shown. H, Simplified description of conditional Rbpjfl/fl construct. I–K, BMDMs from Rbpjfl/fl mice stimulated with 1 mg/mL LPS for 6 hours (I) or overnight (J), or with IL4 for 72 hours (K) with or without previous CRE treatment. I, Relative mRNA expression levels of M1 and M2 marker genes determined by qRT-PCR (n ¼ 6). Mean þ SD is shown. J, Flow cytometry analysis showing percentage of iNOSþ and MRC1þ cells in F4/80þ BMDMs (n ¼ 4). Mean þ SD þ þ is shown. K, Representative histogram showing relative fluorescence intensity of MRC1 on CD11b F4/80 cells upon CRE and IL4. , P < 0.05; , P < 0.005.

5002 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

validated for protein level of iNOS and MRC1 by flow cytometry staining for their coexpression pattern in FKP;Lyz2;N2IC tumors, þ (Fig. 2G). further confirmed the selective Notch activation in CD11b It is noteworthy that IL1b was upregulated by CRE alone myeloid cells (Fig. 3F). treatment (Fig. 2D and F). However, the induced IL1b level by We next addressed the expression of the different Notch recep- CRE alone is significantly lower when compared with that tors. Although Notch2 is the target in R26LSL-N2IC mice, ablation of of CRE plus LPS treatment (Fig. 2F). Besides, the expression Rbpj potentially affects other Notch family members. We thus of other M1-associated genes (e.g., IL6, IL12, iNos) induced by analyzed the constitutive expression of Notch 1–4inFKP tumors. CRE alone is generally subtle. Although we cannot exclude the IHC analysis revealed the expression of Notch 1 and 2, but not possibility that NLS-TAT-CRE exposure indeed might induce Notch 3 and 4 in FKP tumors (Supplementary Fig. S3B). Subse- changes in M1- and/or M2-associated genes, the magnitudes of quent coexpression analysis by immunofluorescence staining differences induced upon LPS and IL4 treatment are at least showed that Notch1 expression was largely found in tumor cells, þ several folds greater than CRE alone. Because the induction but not in CD11b myeloid cells (Supplementary Fig. S3C). of M1 and M2-associated genes is clearly different between Besides, Notch1 expression was similar in either FKP, FKP;Lyz2; N2IC-andRbpj-targeted cells, it is deduced that Notch Rbpj or FKP;Lyz2;N2IC tumors (Supplementary Fig. S3B and S3C), modulation is the dominant factor counteracting IL4-induced suggesting that Notch1 is not expressed in myeloid cells M2 polarization. and therefore not subject to myeloid-specific modulation in our system. Rbpj knockout blocks LPS-induced M1 polarization in vitro Notably, Notch2 was found to be highly coexpressed with Next, we investigated the role of endogenous Notch signaling CD11b, that is, in myeloid cells. As shown in Fig. 3G, modulation fl fl for M1 or M2 macrophage polarization. Rbpj / construct allows of Notch2 is a target of the genetic intervention, as an increase in þ CRE-dependent ablation of canonical Notch signaling (Fig. 2H). Notch2 was mainly found in CD11b cells in FKP;Lyz2;N2IC fl fl BMDMs from Rbpj / mice were treated analogously to those from mice, whereas it was largely reduced in FKP;Lyz2;Rbpj. In fact, our R26LSL-N2IC mice. LPS-induced M1 gene expression was signifi- finding is supported by previous publication that Notch2 is cantly reduced in Rbpj KO macrophages (Fig. 2I). Moreover, fundamental for myeloid cell differentiation (42, 43). LPS-dependent downregulation of Jmjd3, a critical gene for M2 Importantly, we observed a significant survival benefitinLyz2; polarization, was retained in Rbpj KO BMDMs (Fig. 2I). Flow N2IC mice when compared with Lyz2;Rbpj mice in both FKP and cytometric analysis revealed that LPS-induced iNOS expression FKPhet models with deletion of both or only one Tp53 allele, was also downregulated in Rbpj KO macrophages at protein level respectively (Fig. 3H and I), although statistical significance was (Fig. 2J). However, IL4-induced M2 polarization in terms of not reached when compared with FKP controls. MRC1 protein expression was not affected by Rbpj ablation (Fig. 2K). These findings suggest that canonical Notch signaling Notch activation reprograms M2 TAMs in vivo is required for robust M1 polarization. Next, we investigated the effect of Notch modulation on the immune systems in vivo. First, mice were examined for systemic Genetic Notch modulation in myeloid cells using in vivo dual effect of the Lyz2-Cre–mediated Notch manipulation on recombinase system immune landscapes in bone marrow, blood, and spleen. No To validate the regulatory role of Notch in M2 polarization in significant change was observed among Notch activation vivo, a myeloid-targeting Cre strain (Lyz2wt/Cre) was crossed to (Lyz2;N2IC), blockage (Lyz2;Rbpj), and wild-type mice in fl fl R26wt/LSL-N2IC and Rbpj / mice. Lyz2wt/Cre;R26wt/LSL-N2IC (Lyz2; all myeloid and lymphoid subpopulations (Supplementary fl fl N2IC) and Lyz2wt/Cre; Rbpj / (Lyz2;Rbpj) lines were then interbred Fig. S4A–S4C). with Pdx1-Flp;Kraswt/FSF-G12D;p53frt/frt (FKP) mice (Fig. 3A and B). We next performed a detailed characterization of tumors from FKP mice develop and succumb to PDAC highly similar to the FKP;Lyz2;N2IC, FKP;Lyz2;Rbpj, and FKP control mice and CKP model as reported previously (33). Phenotypic characteri- observed that Ki67 expression significantly decreased, whereas zation of FKP tumors was performed showing comparable his- cleaved caspase-3 increased in FKP;Lyz2;N2IC tumors (Fig. 4A), tology and biology of tumor cells, as well as immune profile as suggesting reduced proliferation and increased apoptosis in those of CKP tumors (Supplementary Fig. S3A). Lyz2-Cre expres- tumor cells upon myeloid-targeted Notch activation. Movat's sion was traced with the R26LSL-tdTomato reporter strain to quantify pentachrome staining showed that stromal content was also recombination efficiency in myeloid subsets. Approximately 80% reduced in FKP;Lyz2;N2IC group (Fig. 4A). The opposite trend þ of CD11b myeloid cells were recombined by Lyz2-Cre in bone is observed for the above findings in FKP;Lyz2;Rbpj tumors. It is marrow and spleen (Fig. 3C). Among CD11b cells, there is less noteworthy that collagen (yellowish stain), which was rarely than 10% positivity for tdTomato expression. This population observed in FKP control tumor (Supplementary Fig. S3A), was þ might thus reflect CD11b CD11c dendritic cells that are derived increased in FKP;Lyz2;Rbpj tumor, indicating rearrangement of from myeloid cell precursors (41). To confirm myeloid-specific stromal content. IHC staining was performed to confirm the Notch downstream activation in Lyz2;N2IC, bone marrow of collagen levels in the tumors, and consistently, there were signif- wild-type, Lyz2;N2IC, and Lyz2;Rbpj mice was isolated and sorted icantly higher levels of collagen in FKP;Lyz2;Rbpj tumor when according to CD11b and hCD2 expression for RNA extraction compared with FKP control and FKP;Lyz2;N2IC group (Supple- (Fig. 3D). Real-time qPCR revealed a strong transcriptional upre- mentary Fig. S4D). gulation of the Notch target gene Hes1 in Lyz2;N2IC bone To investigate any alteration in immune landscape upon marrow, indicating that Notch was Lyz2-Cre dependently activat- myeloid-specific Notch modulation, tumors of end-stage FKP; ed, whereas wild-type and Lyz2;Rbpj mice both showed low levels Lyz2;N2IC and FKP;Lyz2;Rbpj mice were characterized for infil- þ of Hes1 expression in CD11b bone marrow cells (Fig. 3E). IHC trating leukocyte subsets. Flow cytometric analysis showed a slight þ þ staining for hCD2 and CD11b, as well as immunofluorescence increase in CD3 T cells and CD19 B cells in FKP;Lyz2;N2IC

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 5003

Cheung et al.

5004 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

mice, although statistical significance was not reached (Fig. 4B). were significantly altered upon Notch modulation in antigen þ CD11b myeloid cells remained the predominant population in processing and MHC pathways. Indeed, these MHC class infiltrating leukocytes in both models. We observed no significant II–related genes were all upregulated for approximately 2- to changes in overall MDSC and TAM proportions, and TAMs 3-fold (log2 scale) in FKP;Lyz2;N2IC tumors (Supplementary þ comprised the majority of CD11b cells (Fig. 4B). However, we Table S6). For chemokines and receptors pathway, Ccl24, a strong þ found a significant reduction of M2 TAMs (iNOS MRC1 )in chemotactic factor for resting T cells (44), was significantly upre- FKP;Lyz2;N2IC tumors when compared with FKP;Lyz2;Rbpj gulated with the greatest fold increase (5-fold in log2 scale) in FKP; (Fig. 4B). Multiplexed immunofluorescent staining for MDSCs Lyz2;N2IC. We next validated findings from NanoString analysis supported the FACS results by showing a slight, but not signif- at protein levels by IHC. Consistent with NanoString analysis, we þ þ þ icant, decrease in G-MDSCs in FKP;Lyz2;N2IC mice, while the observed significantly higher levels of CD3 , CD4 , and CD8 levels of M-MDSCs remained low and there was no change among cells in FKP;Lyz2;N2IC tumors (Fig. 5E). In addition, Eomes and the three groups (Supplementary Fig. S4E). Besides, IHC staining Lag3, markers for both activation and exhaustion of T cells echoed the findings by demonstrating prominent reduction in (45, 46), were among the most significantly altered genes in MRC1 expression in FKP;Lyz2;N2IC tumors (Fig. 4C). Subsequent FKP;Lyz2;N2IC as revealed by NanoString analysis (Supplemen- multiplexed staining for hCD2, CD11b, and MRC1 was per- tary Table S6). Their protein levels were also shown to be formed in FKP;Lyz2;N2IC tumors to assess the M2 phenotype of significantly higher in FKP;Lyz2;N2IC (Fig. 5E). To verify whether þ þ the CD11b hCD2 cells. As shown in Supplementary Fig. S4F, NK cells also contributed the increase in Eomes expression, we MRC1 expression did not show similar pattern to that of hCD2, measured infiltrating NK cells in the tumors and found that NK þ suggesting that hCD2 cells were largely MRC1 negative. The M1 cells were significantly reduced in FKP;Lyz2;N2IC when compared TAM marker iNOS was significantly upregulated in FKP;Lyz2; with FKP;Lyz2;Rbpj (Supplementary Fig. S5A). Therefore, the N2IC tumors, although the level was relatively low (Fig. 4C). increase in Eomes in FKP;Lyz2;N2IC tumors should be largely T cell dependent. Association of myeloid-specific Notch modulation with Next, we validated the markers for antigen presentation as immune profiles in tumor microenvironment revealed by NanoString analysis. IHC staining for CD11c, MHCII, Next, we characterized and compared the immune landscapes and CD80 in FKP;Lyz2;Rbpj, FKP;Lyz2;N2IC and FKP control of FKP;Lyz2;N2IC (n ¼ 7) and FKP;Lyz2;Rbpj (n ¼ 9) tumors using tumors were performed as their genes, CD11c (itgax), CD80, the NanoString PanCancer Immune Panel. The 35 genes most MHCII (e.g., ciita, H2-DMb1, H2-Eb1), were among the most regulated upon Notch modulation are listed in Supplementary significantly upregulated genes upon Notch modulation Table S6 and plotted in the heatmap (Fig. 5A). NanoString (Fig. 5A; Supplementary Table S6). Consistent with NanoString þ þ advanced analysis revealed an increase in infiltrating T cells in analysis, IHC staining showed increase in CD11c , MHCII , and þ þ FKP;Lyz2;N2IC tumors. Specifically, CD4 T cells and exhausted CD80 cells in N2IC-targeted mice, whereas a reduction in FKP; þ CD8 cells significantly increased in FKP;Lyz2;N2IC tumors Lyz2;Rbpj group was observed (Supplementary Fig. S5A). Multi- (Fig. 5B). Note that the cell scores indicate the relative abundance plexed immunofluorescent staining of CD11c with the human þ of immune subsets out of total TILs or total T cells. Consistently, CD2 reporter protein in FKP;Lyz2;N2IC showed that CD11c cells þ flow cytometric analysis also revealed increased CD3 (Fig. 4E), are mostly, if not all, hCD2 negative (Supplementary Fig. S5B). þ þ CD4 , and CD8 T cells (Fig. 5C) in FKP;Lyz2;N2IC, although Subsequent costaining of CD11b, CD11c, MHCII, and CD80 in statistical significance was not reached, likely due to small sample FKP;Lyz2;N2IC showed that upregulation of MHCII and CD80 is þ size (n ¼ 3; Fig. 5C). largely expressed by CD11c cells (Supplementary Fig. S5C). The þ þ Besides, immune pathway analysis by NanoString showed presence of hCD2 CD11c cells might be due to the fact that þ higher scores for antigen processing, MHC, IFN, senescence, some CD11c cells are derived from myeloid cell precursors, as chemokines and receptors, inflammation, and apoptosis path- reported by Clausen and colleagues (41). Regarding the cytokine ways in FKP;Lyz2;N2IC tumors (Fig. 5D). This analysis revealed profile, we observed an increase in the M1-associated cytokines that MHC class II–related genes, for example, Lag3, Ciita, IFNg, IL12p70, and TNFa, whereas the M2-associated cytokines H2-DMb1, and H2-Eb1, comprised the majority of genes that CXCL1 and TGFb were decreased in FKP;Lyz2;N2IC mice

Figure 3. Lyz2-Cre directs genetic Notch activation to myeloid cells. A, Mouse crossing strategy. Pdx1-Flp;Kraswt/FSF-G12D;p53frt/frt (FKP) mice crossed to Lyz2;Rbpj or Lyz2;N2IC strains to generate FKP;Lyz2;Rbpj and FKP;Lyz2;N2IC mice, respectively. B, Oncogenic KrasG12D-driven pancreatic tumorigenesis (FKP) and myeloid-specific manipulation of Notch signaling were concomitantly genetically induced. C, The R26LSL-tdTomato reporter mouse line was used to visualize Lyz2-Cre expression and recombination activity in vivo. tdTomatoþ fractions in CD11bþ and CD11c cells isolated from bone marrow (BM) and spleen (SP) were quantified by flow cytometry (n ¼ 3). Mean þ SD is shown. D, Bone marrow–derived cells of wild-type, Lyz2;N2IC,andLyz2;Rbpj mice were sorted based on hCD2 and CD11b expression, and mRNA was extracted from the sorted populations (red gates). Lower gate, CD11bþhCD2; upper gate, CD11bþhCD2þ. E, Specific Hes1 expression in sorted CD11bþhCD2 and CD11bþhCD2þ cells was assessed by qRT-PCR. F, Left, IHC staining for CD11b, hCD2, and isotype control on the tumor of FKP; Lyz2;N2IC tumor. Magnification, 100; 200. Scale bar, 50 mm. Right, immunofluorescence staining for coexpression of CD11b (green) and hCD2 (red) on the tumor of FKP;Lyz2;N2IC tumor. DAPI (blue), nuclei. Magnification, 25; 200. Scale bar, 100 mm. G, Immunofluorescence staining for coexpression of CD11b (green) and Notch2 (red) on the tumor of FKP, FKP;Lyz2;Rbpj,andFKP;Lyz2;N2IC tumors. DAPI (blue), nuclei. Magnification, 25; 200. Scale bar, 100 mm. H and I, Kaplan– Meier plot showing the survival of FKP;Lyz2;Rbpj, FKP;Lyz2;N2IC,andFKP control mice in FKP (H)andFKPhet (I) models. H, Median survival: FKP;Lyz2;Rbpj, 67 days (n ¼ 24); FKP;Lyz2;N2IC, 78 days (n ¼ 11); FKP,70days(n ¼ 19). Log-rank test: FKP;Lyz2;Rbpj versus FKP;Lyz2;N2IC, P ¼ 0.014; FKP;Lyz2;Rbpj versus FKP, P ¼ 0.482; FKP;Lyz2;N2IC versus FKP, P ¼ 0.095. I, Median survival: FKPhet;Lyz2;Rbpj, 147 days (n ¼ 17); FKPhet;Lyz2;N2IC, 186.5 days (n ¼ 8); FKPhet, 172 days (n ¼ 24). Log-rank test: FKP;Lyz2;Rbpj versus FKP;Lyz2;N2IC, P ¼ 0.013; FKP;Lyz2;Rbpj versus FKP, P ¼ 0.184; FKP;Lyz2;N2IC versus FKP, P ¼ 0.317. , P < 0.05 when comparing between groups denoted by horizontal bars.

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 5005

Cheung et al.

Figure 4. Notch signaling antagonizes M2 polarization in PDAC TAMs. A, Movat's pentachrome staining, IHC staining of Ki67, and cleaved caspase-3 in FKP, FKP;Lyz2;Rbpj,and FKP;Lyz2;N2IC tumors (n 5/group). Magnification, 25; 200. Scale bar, 100 mm. Bottom, percentage of positive cells or area (for Movat's pentachrome staining only) as the average of 5 fields from each mouse (objective magnification, 10). Mean þ SD is shown. B, Tumors of end-stage FKP;Lyz2;Rbpj and FKP;Lyz2;N2IC mice were digested into desegregated cells and analyzed by flow cytometry. Live CD45þ cells were gated as viable leukocytes for subsequent þ þ þ þ hi immune subpopulation characterization: T cells (CD3 ), B cells (CD19 ), and myeloid cells (CD11b ); M-MDSCs (CD11b F4/80 /loGR1 /loLy6C ), G-MDSCs þ hi þ þ (CD11b F4/80 GR1 ), and TAM (CD11b F4/80 GR1 /lo). TAMs were further dissected into subpopulations based on iNOS and MRC1 expression. Mean þ SD is shown. C, IHC staining for CD11b, F4/80, MRC1, and iNOS in FKP;Lyz2;Rbpj and FKP;Lyz2;N2IC tumors (n 5/group). Magnification, 25; 200. Scale bar, 100 mm. Bottom, percentage of positive cells as the average of 5 fields (n 5/group; objective magnification, 10). Mean þ SD is shown. , P < 0.05; , P < 0.01.

5006 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

Figure 5. Association of myeloid-specific Notch modulation with immune landscapes in tumor microenvironment. A, B, and D, Profile of immune-related expression signatures in FKP;Lyz2;Rbpj (n ¼ 9) and FKP;Lyz2;N2IC (n ¼ 7) tumors determined by the NanoString PanCancer Immune Profiling Panel. A, Heatmap with hierarchical clustering for genes with at least 2-fold change up or down with P < 0.05 as cutoff. Significant upregulation is shown in red and downregulation in green. B, The cell-type–specific scores of T cells and exhausted CD8þ T cells (relative to total tumor-infiltrating cells), and CD4þ T cells (relative to total CD3þ Tcells) were calculated by PanCancer Immune Profiling Advanced Analysis as described in Materials and Methods. C, Flow cytometric analysis showing the percentage of CD4þ T cells (CD3þCD4þCD8) and CD8þ T cells (CD3þCD4CD8þ) of total leukocytes (CD45þ)inFKP;Lyz2;Rbpj and FKP; Lyz2;N2IC tumors (n ¼ 3/group). D, Pathway scores of antigen processing, MHC, IFN, senescence, chemokines and receptors, inflammation, and apoptosis were calculated by PanCancer Immune Profiling Advanced Analysis as described in Materials and Methods. E, IHC staining of CD3, CD4, CD8, Eomes, and LAG3 in FKP;Lyz2;Rbpj and FKP;Lyz2;N2IC tumors (n 5/group). Magnification, 25; 200. Scale bar, 100 mm. Right, percentage of positive cells as the average of 5 fields (n 5/group; objective magnification, 10). Mean þ SD is shown. , P < 0.05; , P < 0.005; , P < 0.001.

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 5007

Cheung et al.

(Supplementary Fig. S5D). It is noteworthy that serum TGFb statistically significant, would be consistent with this effect levels were also decreased in the FKP;Lyz2;N2IC group, suggesting (Fig. 4B; Supplementary Fig. S4E). Thus, we cannot rule out a systemic change in this cytokine. However, no statistical signif- the possibility that Notch-induced MDSC differentiation icance was reached in the above cytokine profiling due to the small also contributed to the observed alterations in the tumor sample size. microenvironment. Further investigation will be required In addition to the above-mentioned cell types and pathways, to further dissect the roles of MDSCs and TAMs in modulating no significant change was observed for the infiltration of immune immunosuppression. cell types including B cells, mast cells, neutrophils (Supplemen- Analysis of immune landscape of FKP;Lyz2;N2IC and FKP;Lyz2; tary Fig. S5E), and pathways, for example, TNF, NK function, and Rbpj tumors by NanoString showed that Notch-induced M2 innate immunity (Supplementary Fig. S5F). reduction was significantly associated with gene expression signatures of increased antigen processing and presentation, fi Discussion in ltrating T cells, and IFN pathway. Intriguingly, a strong expression of exhausted T-cell signature was observed in FKP;Lyz2;N2IC OneofthedistinctivefeaturesofPDACisthatthemalignant mice, in which Eomes, Lag3, and PD-1 were expressed at high epithelial cells often account for only a minority of tumor mass, levels. Although described as exhaustion markers for T cells, they whereas the desmoplastic stroma and other nontumor cells are in many ways considered as T-cell activation markers (45, 46, constitute up to 80% (47). Currently, most targeting 57). Upregulation of these markers may suggest that these cells approaches focus on aberrant signaling of tumor cells, while have undergone priming and were consequently activated. One the effect on other cellular compartments remains elusive given key feature of T-cell exhaustion is the continuous exposure to the lack of comprehensive model systems. As such, targeting of antigen rather than acutely terminated or intermittent exposure Notch signaling has been disappointing clinically in PDAC (58). Here, we observed that upon M2 reduction, antigen proces- despite supportive preclinical evidence. However, in more sing and presentation was significantly enhanced, which might at complex disease models, the role of Notch signaling has been least partially explain the restored T-cell activation and potentially controversial in PDAC, exerting both pro- and antitumorigenic the survival benefitofFKP;Lyz2;N2IC mice. This is supported effects (48). by the strong correlation between the mRNA levels of Eomes, In this study, we used a genetic approach to modulate Notch Lag3, and PD-1, with antigen processing/presentation–related signaling specifically in myeloid cells and characterized the genes that are highly upregulated upon Notch activation in consequent effects on the immune landscape in spontaneous myeloid cells. endogenous PDAC in immunocompetent mice. In line with In addition to T-cell infiltration and activation, another inter- previous findings, tumor-promoting M2 TAMs were shown to esting impact of myeloid-specific Notch modulation is antigen predominate the TILs (49, 50). A significant amount of M2 TAMs presentation. NanoString analysis and subsequent IHC staining was observed in preneoplastic lesions, and the level increased demonstrated augmented antigen presentation in FKP;Lyz2;N2IC along tumor progression, suggesting that M2 TAMs participate not tumors. Upregulated MHCII and CD80 were predominantly þ only in PDAC progression, but also in tumor formation (51–53). expressed by CD11c dendritic cells, which are mostly hCD2 þ Here, we demonstrated a tumor-suppressive role of Notch negative. One possible explanation for increased CD11c signaling in myeloid cells in PDAC. Upon Notch activation in dendritic cells may be an altered cytokine and chemokine profile myeloid cells, M2 TAMs were significantly reduced, while antigen in the tumor microenvironment. Although not statistically presentation and cytotoxic T-cell activity were restored, and significant, cytokine profiling revealed increased cytotoxic importantly, survival of mice with spontaneous PDAC was cytokines IFNg, IL12p70, and TNFa, and decreased tumor- significantly improved. Our findings therefore may help explain promoting cytokines CXCL1 and TGFb in FKP;Lyz2;N2IC tumors the limited efficacy of targeting Notch signaling in PDAC as (Supplementary Fig. S5D). Besides, a systemic change in TGFb previously reported (13, 14). was observed in FKP;Lyz2;N2IC mice, as illustrated by decreased Earlier studies reporting the repolarization of macrophages in serum TGFb level. It is notable that TGFb is crucial for inducing experimental PDAC demonstrated beneficial outcome when M2 M2 phenotype and inhibiting dendritic cell maturation and polarization was antagonized either genetically or by low-dose activation (59). A reduction in TGFb might at least partially þ irradiation (54, 55). These studies, however, were conducted explain the increased CD11c dendritic cells with higher MHCII either by tumor transplantation or transgenic mouse models, and CD80 expression levels in FKP;Lyz2;N2IC cohort. In this which do not recapitulate seminal features of human PDAC as regard, it can be speculated that the application of Notch inhibi- faithfully as spontaneous KrasG12D-driven PDAC mouse models tors in clinical settings might lead to suppressed antigen presen- do. We thus employed a novel dual-recombinase system to tation, which potentially compromises the efficacy of other genetically induce both KrasG12D-driven pancreatic tumorigenesis immunotherapies. However, further investigation is needed to and myeloid-specific modulation of Notch signaling. In our Lyz2- comprehensively characterize the impact of the Notch-induced CRE model, Notch signaling was genetically targeted in a myeloid myeloid polarization on the complex antigen presentation cell–specific manner. It is noteworthy that although TAMs machinery. represent the majority (50%–60%) of intratumoral myeloid The current study may serve as proof of concept for genetic cells in our PDAC model, MDSCs also account for a significant targeting of myeloid subsets in complex models of immunocom- proportion (20%–30%). Indeed, recent studies have shown that petent endogenous PDAC. The dual recombinase-based approach Notch signaling can regulate MDSC differentiation. Blockage offers a platform to assess their contribution in PDAC progression of Notch signaling promoted the differentiation and expansion and immune-based approaches. Our findings on the antitumori- of G-MDSCs both in vitro and in vivo (29, 56). Our finding of a genic role of Notch signaling specifically in myeloid cells call for reduction in G-MDSCs upon Notch activation, although not attention on the need to dissect the differential roles of signaling

5008 Cancer Res; 78(17) September 1, 2018 Cancer Research

Myeloid-Speciﬁc Notch Modulation in Pancreatic Cancer

pathways in different cellular components within the tumor Administrative, technical, or material support (i.e., reporting or organizing microenvironment. The dual-recombinase system is a useful data, constructing databases): P.F. Cheung, C. Neander, A. Bazarna, model system for dissecting the complex network among K. Savvatakis, S.-T. Liffers Study supervision: P.F. Cheung, J.T. Siveke tumor and stromal components to validate nontumor-targeting strategies in PDAC, a disease in high need for better treatment Acknowledgments approaches. The authors would like to thank M. Schmid-Supprian for helpful discussions and providing recombinant CRE protein; U. Zimber-Strobel for providing Disclosure of Potential Conflicts of Interest R26LSL-N2IC mice; H. Nakhai for floxed Rbpj and Ptf1aCre mice; T. Jacks and LSL-G12D No potential conflicts of interest were disclosed. D.A. Tuveson for Kras mice; and A. Berns for floxed p53 mice. We thank N. Bielefeld, R. Hillermann, and S. Sch€afers for excellent technical assistance and Authors' Contributions the International Max Planck Research School for Molecular Life Sciences (IMPRS) for providing educational and financial support to F. Neff. J.T. Siveke Conception and design: P.F. Cheung, F. Neff, J.T. Siveke had been awarded for European Union's Seventh Framework Programme for Development of methodology: P.F. Cheung, F. Neff, C. Neander, A. Bazarna, research, technological development and demonstration (FP7/CAM-PaC) K. Savvatakis, S.-T. Liffers, D. Saur, J.T. Siveke under grant agreement no. 602783, the German Cancer Consortium (DKTK), Acquisition of data (provided animals, acquired and managed patients, and the Deutsche Forschungsgemeinschaft (DFG, SI1549/1-1). provided facilities, etc.): P.F. Cheung, F. Neff, K. Savvatakis, S.-T. Liffers, K. Althoff, C.-L. Lee, E.J. Moding, D.G. Kirsch, D. Saur, M. Trajkovic-Arsic, M.F. Heikenwalder The costs of publication of this article were defrayed in part by the payment of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, page charges. This article must therefore be hereby marked advertisement in computational analysis): P.F. Cheung, F. Neff, C. Neander, A. Bazarna, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S.-T. Liffers, A.V. Bazhin, M.F. Heikenwalder, J.T. Siveke Writing, review, and/or revision of the manuscript: P.F. Cheung, F. Neff, Received January 7, 2018; revised April 20, 2018; accepted May 22, 2018; E.J. Moding, D.G. Kirsch, A.V. Bazhin, M.F. Heikenwalder, J.T. Siveke published first May 29, 2018.

References 1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. 16. Bayne LJ, Beatty GL, Jhala N, Clark CE, Rhim AD, Stanger BZ, et al. Cell 2011;144:646–74. Tumor-derived granulocyte-macrophage colony-stimulating factor reg- 2. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. ulates myeloid inflammation and T cell immunity in pancreatic cancer. Nature 2008;454:436–44. Cancer Cell 2012;21:822–35. 3. Chu GC, Kimmelman AC, Hezel AF, DePinho RA. Stromal biology of 17. Nowell CS, Odermatt PD, Azzolin L, Hohnel S, Wagner EF, Fantner GE, pancreatic cancer. J Cell Biochem 2007;101:887–907. et al. Chronic inflammation imposes aberrant cell fate in regenerating 4. Neesse A, Krug S, Gress TM, Tuveson DA, Michl P. Emerging concepts in epithelia through mechanotransduction. Nat Cell Biol 2016;18: pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther 168–80. 2013;7:33–43. 18. Dumortier A, Durham AD, Di Piazza M, Vauclair S, Koch U, Ferrand G, et al. 5. Stromnes IM, DelGiorno KE, Greenberg PD, Hingorani SR. Stromal reen- Atopic dermatitis-like disease and associated lethal myeloproliferative gineering to treat pancreas cancer. Carcinogenesis 2014;35:1451–60. disorder arise from loss of Notch signaling in the murine skin. PLoS One 6. Koch U, Radtke F. Notch signaling in solid tumors. Curr Top Dev Biol 2010;5:e9258. 2010;92:411–55. 19. Demehri S, Liu Z, Lee J, Lin MH, Crosby SD, Roberts CJ, et al. Notch- 7. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio-Donahue deficient skin induces a lethal systemic B-lymphoproliferative disorder by CA, et al. Notch mediates TGF alpha-induced changes in epithelial differ- secreting TSLP, a sentinel for epidermal integrity. PLoS Biol 2008;6:e123. entiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565–76. 20. Demehri S, Turkoz A, Manivasagam S, Yockey LJ, Turkoz M, Kopan R. 8. Plentz R, Park JS, Rhim AD, Abravanel D, Hezel AF, Sharma SV, et al. Elevated epidermal thymic stromal lymphopoietin levels establish an Inhibition of gamma-secretase activity inhibits tumor progression in a antitumor environment in the skin. Cancer Cell 2012;22:494–505. mouse model of pancreatic ductal adenocarcinoma. Gastroenterology 21. Di Piazza M, Nowell CS, Koch U, Durham AD, Radtke F. Loss of cutaneous 2009;136:1741–9e6. TSLP-dependent immune responses skews the balance of inflammation 9. Blackburn SD, Shin H, Freeman GJ, Wherry EJ. Selective expansion of a from tumor protective to tumor promoting. Cancer Cell 2012;22:479–93. subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad 22. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor Sci U S A 2008;105:15016–21. microenvironments. Cancer Res 2006;66:605–12. 10. Murtaugh LC, Stanger BZ, Kwan KM, Melton DA. Notch signaling controls 23. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polar- multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 2003; ization: tumor-associated macrophages as a paradigm for polarized M2 100:14920–5. mononuclear phagocytes. Trends Immunol 2002;23:549–55. 11. Hanlon L, Avila JL, Demarest RM, Troutman S, Allen M, Ratti F, et al. 24. Beatty GL, Winograd R, Evans RA, Long KB, Luque SL, Lee JW, et al. Notch1 functions as a tumor suppressor in a model of K-ras-induced Exclusion of T cells from pancreatic carcinomas in mice is regulated by pancreatic ductal adenocarcinoma. Cancer Res 2010;70:4280–6. Ly6C(low) F4/80(þ) extratumoral macrophages. Gastroenterology 12. Mazur PK, Einwachter H, Lee M, Sipos B, Nakhai H, Rad R, et al. Notch2 is 2015;149:201–10. required for progression of pancreatic intraepithelial neoplasia and devel- 25. Knudsen E, Vail P, Balaji U, Ngo H, Botros IW, Makarov V, et al. Strati- opment of pancreatic ductal adenocarcinoma. Proc Natl Acad Sci U S A fication of pancreatic ductal adenocarcinoma: combinatorial genetic, stro- 2010;107:13438–43. mal, and immunological markers. Clin Cancer Res 2017;23:4429–40. 13. Ott PA, Hodi FS, Robert C. CTLA-4 and PD-1/PD-L1 blockade: new 26. Hutcheson J, Balaji U, Porembka MR, Wachsmann MB, McCue PA, Knud- immunotherapeutic modalities with durable clinical benefit in melanoma sen ES, et al. Immunologic and metabolic features of pancreatic ductal patients. Clin Cancer Res 2013;19:5300–9. adenocarcinoma define prognostic subtypes of disease. Clin Cancer Res 14. Anderson AC. Tim-3: an emerging target in the cancer immunotherapy 2016;22:3606–17. landscape. Cancer Immunol Res 2014;2:393–8. 27. Di Caro G, Cortese N, Castino GF, Grizzi F, Gavazzi F, Ridolfi C, et al. Dual 15. Demehri S, Turkoz A, Kopan R. Epidermal Notch1 loss promotes skin prognostic significance of tumour-associated macrophages in human tumorigenesis by impacting the stromal microenvironment. Cancer Cell pancreatic adenocarcinoma treated or untreated with chemotherapy. Gut 2009;16:55–66. 2016;65:1710–20.

www.aacrjournals.org Cancer Res; 78(17) September 1, 2018 5009

Cheung et al.

28. Yoshikawa K, Mitsunaga S, Kinoshita T, Konishi M, Takahashi S, Gotohda inhibitors of two distinct classes of myeloid progenitors. J Exp Med N, et al. Impact of tumor-associated macrophages on invasive ductal 1997;185:1163–72. carcinoma of the pancreas head. Cancer Sci 2012;103:2012–20. 45. Buggert M, Tauriainen J, Yamamoto T, Frederiksen J, Ivarsson MA, 29. Cheng P, Kumar V, Liu H, Youn JI, Fishman M, Sherman S, et al. Effects of Michaelsson J, et al. T-bet and Eomes are differentially linked to the notch signaling on regulation of myeloid cell differentiation in cancer. exhausted phenotype of CD8þ T cells in HIV infection. PLoS Pathog Cancer Res 2014;74:141–52. 2014;10:e1004251. 30. Wang YC, He F, Feng F, Liu XW, Dong GY, Qin HY, et al. Notch signaling 46. Grosso JF, Goldberg MV, Getnet D, Bruno TC, Yen HR, Pyle KJ, et al. determines the M1 versus M2 polarization of macrophages in antitumor Functionally distinct LAG-3 and PD-1 subsets on activated and chronically immune responses. Cancer Res 2010;70:4840–9. stimulated CD8 T cells. J Immunol 2009;182:6659–69. 31. Xu H, Zhu J, Smith S, Foldi J, Zhao B, Chung AY, et al. Notch-RBP-J signaling 47. Farrow B, Albo D, Berger DH. The role of the tumor microenvironment in regulates the transcription factor IRF8 to promote inflammatory macro- the progression of pancreatic cancer. J Surg Res 2008;149:319–28. phage polarization. Nat Immunol 2012;13:642–50. 48. Nowell CS, Radtke F. Notch as a tumour suppressor. Nat Rev Cancer 32. Mazur PK, Siveke JT. Genetically engineered mouse models of pancreatic 2017;17:145–59. cancer: unravelling tumour biology and progressing translational oncol- 49. Mitchem JB, Brennan DJ, Knolhoff BL, Belt BA, Zhu Y, Sanford DE, et al. ogy. Gut 2012;61:1488–500. Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, 33. Schonhuber N, Seidler B, Schuck K, Veltkamp C, Schachtler C, Zukowska relieves immunosuppression, and improves chemotherapeutic responses. M, et al. A next-generation dual-recombinase system for time- and host- Cancer Res 2013;73:1128–41. specific targeting of pancreatic cancer. Nat Med 2014;20:1340–7. 50. Ino Y, Yamazaki-Itoh R, Shimada K, Iwasaki M, Kosuge T, Kanai Y, et al. 34. Hampel F, Ehrenberg S, Hojer C, Draeseke A, Marschall-Schroter G, Immune cell infiltration as an indicator of the immune microenvironment Kuhn R, et al. CD19-independent instruction of murine marginal zone of pancreatic cancer. Br J Cancer 2013;108:914–23. B-cell development by constitutive Notch2 signaling. Blood 2011;118: 51. Liou GY, Bastea L, Fleming A, Doppler H, Edenfield BH, Dawson DW, et al. 6321–31. The presence of interleukin-13 at pancreatic ADM/PanIN lesions alters 35. Nakhai H, Siveke JT, Klein B, Mendoza-Torres L, Mazur PK, Algul H, et al. macrophage populations and mediates pancreatic tumorigenesis. Cell Rep Conditional ablation of Notch signaling in pancreatic development. 2017;19:1322–33. Development 2008;135:2757–65. 52. Zhang Y, Yan W, Mathew E, Kane KT, Brannon A, Adoumie M, et al. 36. Heger K, Kober M, Riess D, Drees C, de Vries I, Bertossi A, et al. A novel Cre Epithelial-myeloid cell crosstalk regulates acinar cell plasticity and pan- recombinase reporter mouse strain facilitates selective and efficient infec- creatic remodeling in mice. eLife 2017;6:pii:e27388. tion of primary immune cells with adenoviral vectors. Eur J Immunol 53. Zhang Y, Velez-Delgado A, Mathew E, Li D, Mendez FM, Flannagan K, et al. 2015;45:1614–20. Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the 37. Mazur PK, Herner A, Mello SS, Wirth M, Hausmann S, Sanchez-Rivera FJ, establishment of an immunosuppressive environment in pancreatic can- et al. Combined inhibition of BET family proteins and histone deacetylases cer. Gut 2017;66:124–36. as a potential epigenetics-based therapy for pancreatic ductal adenocarci- 54. Gironella M, Calvo C, Fernandez A, Closa D, Iovanna JL, Rosello-Catafau J, noma. Nat Med 2015;21:1163–71. et al. Reg3beta deficiency impairs pancreatic tumor growth by skewing 38. Speiser DE, Ho PC, Verdeil G. Regulatory circuits of T cell function in macrophage polarization. Cancer Res 2013;73:5682–94. cancer. Nat Rev Immunol 2016;16:599–611. 55. Klug F, Prakash H, Huber PE, Seibel T, Bender N, Halama N, et al. Low-dose 39. Kaech SM, Cui W. Transcriptional control of effector and memory CD8þ T irradiation programs macrophage differentiation to an iNOS(þ)/M1 phe- cell differentiation. Nat Rev Immunol 2012;12:749–61. notype that orchestrates effective T cell immunotherapy. Cancer Cell 40. Schmidt-Supprian M, Rajewsky K. Vagaries of conditional gene targeting. 2013;24:589–602. Nat Immunol 2007;8:665–8. 56. Wang SH, Lu QY, Guo YH, Song YY, Liu PJ, Wang YC. The blockage of 41. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene Notch signalling promoted the generation of polymorphonuclear mye- targeting in macrophages and granulocytes using LysMcre mice. Transgenic loid-derived suppressor cells with lower immunosuppression. Eur J Cancer Res 1999;8:265–77. 2016;68:90–105. 42. Bigas A, Martin DI, Milner LA. Notch1 and Notch2 inhibit myeloid 57. Intlekofer AM, Takemoto N, Wherry EJ, Longworth SA, Northrup JT, differentiation in response to different cytokines. Mol Cell Biol 1998; Palanivel VR, et al. Effector and memory CD8þ T cell fate coupled by 18:2324–33. T-bet and eomesodermin. Nat Immunol 2005;6:1236–44. 43. Varnum-Finney B, Halasz LM, Sun M, Gridley T, Radtke F, Bernstein ID. 58. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaus- Notch2 governs the rate of generation of mouse long- and short-term tion. Nat Rev Immunol 2015;15:486–99. repopulating stem cells. J Clin Invest 2011;121:1207–16. 59. Yang L, Pang Y, Moses HL. TGF-beta and immune cells: an important 44. Patel VP, Kreider BL, Li Y, Li H, Leung K, Salcedo T, et al. Molecular and regulatory axis in the tumor microenvironment and progression. Trends functional characterization of two novel human C-C chemokines as Immunol 2010;31:220–7.

5010 Cancer Res; 78(17) September 1, 2018 Cancer Research

Notch-Induced Myeloid Reprogramming in Spontaneous Pancreatic Ductal Adenocarcinoma by Dual Genetic Targeting

Phyllis F. Cheung, Florian Neff, Christian Neander, et al.

Cancer Res 2018;78:4997-5010. Published OnlineFirst May 29, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0052

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2018/05/26/0008-5472.CAN-18-0052.DC1

Cited articles This article cites 59 articles, 21 of which you can access for free at: http://cancerres.aacrjournals.org/content/78/17/4997.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/78/17/4997.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/78/17/4997. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.