Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.

Pancreatic ductal adenocarcinoma (PDAC) is associated with an immunosuppressive tumor microenvironment (TME), which potentially explains the poor response to immunotherapeutic agents including checkpoint inhibitors (1). PDAC TME attracts several immunosuppressive cell types that coordinately act to inhibit the effectiveness of cytotoxic CD8+ T cells, thus attenuating their antitumor function (1, 2). These immune cell types include myeloid-derived suppressor cells, regulatory T cells, and tumor-associated (M2) macrophages (1, 3). M2-macrophages are an abundant immunosuppressive cell type in the PDAC TME (4). In this report, we investigated the mechanism by which PDAC cells regulate M2-macrophage generation (a process called polarization) and its tumor immunosuppression activity.

Macrophages regularly sense their microenvironment and, depending on the inflammatory signal, adopt either an M1 or M2 phenotype (5). M1-macrophages are proinflammatory, whereas M2-macrophages are anti-inflammatory, protumorigenic, and immunosuppressive (5). The density of M2-macrophages in the PDAC TME is highly correlated with a poor prognosis (6). Although the origin of M2-macrophages is not fully understood, experimental evidence suggests that tumor-dependent mechanisms are responsible for macrophage polarization (7). M2-macrophages facilitate tumor invasion, metastasis, and enhance chemoresistance (5, 7). Recent reports suggest that M2-macrophages can suppress the antitumor activity of cytotoxic CD8+ T cells within tumors (8). Reversing the immunosuppressive effects of M2-macrophages offers an attractive therapeutic strategy to enhance host antitumor immunity.

Doublecortin-like kinase 1 (DCLK1) is a marker of tumor stem cells, which identifies the progenitors and cell of origin in inflammation-associated mouse pancreatic cancer (9–11). DCLK1 is overexpressed in the tumors and pancreatic intraepithelial lesions of various pancreatic cancer models (9, 10, 12). Patients with pancreatic cancer with DCLK1-expressing tumors exhibit poor survival compared with patients without DCLK1-expressing tumors (13). DCLK1 inhibition in pancreatic cancer cells blocks tumor cell migration/invasion and results in tumor xenograft growth arrest (14). Recent studies have identified the important function of DCLK1-expressing intestinal tuft cells in regulating the immune response after infection and/or injury (15). Furthermore, it is reported that DCLK1-expressing tuft cells are the major local source of IL17 and COX1/COX2-mediated PGE2 which play crucial roles in regulating proinflammatory molecules and immune cells (16, 17). It is demonstrated that KrasG12D activation in the pancreas is associated with the abnormal generation of pancreatic tuft cells (16), and these cells are identified as progenitor cells and tumor-initiating cells after injury (10). Although earlier studies have suggested the critical role for Dclk1+ cells in pancreatic cancer development after severe injury, their role in the regulation of immune cells for the development of immunosuppressive TME in PDAC has not been established.

Human DCLK1 consists of four primary isoforms, each with a shared kinase domain, driven from two promoter regions termed α and β (Supplementary Fig. S1; ref. 18). The α-promoter drives the expression of isoforms termed α-long (isoform2) and α-short (isoform1), and the β-promoter drives the expression of isoforms termed β-long (isoform4) and β-short (isoform3). However, the potential tumorigenic function and tumor immune regulation of these isoforms are not clear.

In this article, we analyzed the role of DCLK1-isoform2–expressing PDAC cells in macrophage polarization and examined its immunosuppression for tumor progression. Our findings suggest that DCLK1-isoform2 expression in PDAC cells induces polarization of macrophages that contribute to the aggressiveness of PDAC cells and inhibition of cytotoxic T-cell function.

Experimental animals

The KRasLSLG12Dp53LSLR172HPdx1CreRosa26YFP (KPCY) and control mice were described previously (19). Mice were housed under controlled conditions and were monitored regularly. All animal experiments were performed with the approval and authorization from the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center.

Mouse samples

Fixed CD8+ T-cell high (“T-cell high”) and CD8+ T-cell low (“T-cell low”) pancreatic adenocarcinoma tissues from autochthonous mouse model (20) were generously provided by Ben Z. Stanger, MD, Ph.D., of the Perelman School of Medicine, University of Pennsylvania.

Determination of DCLK1-correlated immunosuppressive markers and macrophage polarization markers in The Cancer Genome Atlas PAAD patient data

The pancreatic adenocarcinoma (PAAD) RNA sequencing (RNA-seq) datasets in The Cancer Genome Atlas (TCGA) database were downloaded through the UCSC cancer genome browser, as previously described (21). The corrplot function (R package corrplot) was used to confirm the correlation between the expression levels of DCLK1 and other genes.

Analysis of DCLK1 isoforms expression

TSVdb is a web-based tool (22) that enables to readily access, analyze, and interpret alternative splicing based on TCGA PAAD samples for DCLK1. We interpret DCLK1 isoforms expression variations between solid tumor tissues and normal adjacent tissues. TSVdb is available at http://www.tsvdb.com.

Cell culture

THP-1 cells were purchased in 2018 from the ATCC. THP1 cells were treated with 100 ng/mL of Phorbol-12-myristate-13-acetate (Sigma-Aldrich) for 48 hours. M1-macrophages were then generated by the addition of IFNγ (100 ng/mL). Macrophage polarization was confirmed by M1 and M2 markers expression. Human PDAC cell lines, AsPC-1, and MIA PaCa were obtained from the ATCC in 2017. PDAC cells were grown in DMEM supplemented with 10% FBS (Sigma) at 37°C and 5% CO2. Human CD8+ T cells (TLL-104) were obtained in 2018 from the ATCC and grown in T-cell–specific Iscove's modified Dulbecco's medium. Lentivirus containing human DCLK1isoform2 cDNA sequence was constructed as described previously (23). All cell lines were used within 25 passages. All the cells were confirmed to be negative for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza). AsPC-1 and MIA PaCa2 were infected with lentivirus to overexpress DCLK1-isoform2-RFP fusion protein (AsPC1-DCLK1-isoform2/MIA PaCa2-DCLK1isoform2) or RFP (AsPC1-RFP/MIA PaCa2-RFP) as control and selected with puromycin to establish stable cell lines. Coculture experiments, those that physically interact (juxtacrine): M1-macrophages or M2-macrophages (1 × 105 cells per 6-well plate) were plated in a 6-well plate, and after 24 hours, AsPC1 or MIA PaCa2 or T cells at a concentration of 1 × 105 cells were seeded for coculture. Dual-culture experiments, those that physically separate from one another using transwell plates that allow communication only via soluble factors (paracrine): M1-macrophages or M2-macrophages (1 × 105 cells per 6-well plate) were plated in the 6-well plates, and AsPC1 or MIA PaCa2 or T cells at a concentration of 1 × 105 cells were seeded in the transwell inserts for dual-culture. Coculture experiments, those that physically interact (juxtacrine): M1-macrophages or M2-macrophages (1 × 105 cells per 6-well plate) were plated in a 6-well plate, and after 24 hours, AsPC1 and T cells at a concentration of 1 × 105 cells were seeded for coculture.

siRNA-mediated knockdown of DCLK1

PDAC cells were seeded into 6 cm Petri dishes and allowed to attach overnight. Following attachment, 1 nmol/L of commercially validated siRNA targeting human DCLK1 or 1 nmol/L human scrambled sequence (siSCR) not targeting any known genes (Santa Cruz Biotechnology) were complexed with Lipofectamine 3000 (Invitrogen) and added to the dishes in fresh cell culture MEM medium. After 48 hours, cells were used for further analysis.

Cell proliferation assays

After treatment, 10 μL of TACS MTT Reagent (RND Systems) was added to each well, and the cells were incubated at 37°C until a dark crystalline precipitate became visible in the cells. Then 100 μL of 266 mmol/L NH4OH in DMSO was added to the wells and placed on a plate shaker at low speed for 5 minutes. After shaking, the plate was allowed to incubate for 10 minutes protected from light, and the OD550 for each well was read using a microplate reader. The results were averaged and calculated as percentage cell proliferation.

Clonogenic assay

Reduced growthfactor matrigel was mixed with cell suspensions containing RPMI medium (volume 1:1). A hundred microliters of the mixture were pipetted into 48-well plates at 500 cells/well. After matrigel solidification, a 500 μL RPMI medium with serum supplement was added to each well. The plates were then incubated at 37°C with 5% CO2. The media were refreshed weekly and wells monitored for spheroid formation, as described previously (17, 21, 24–26).

Organoid coculture

Organoids were generated as described in the clonogenic assay. Tumor organoid and immune cell coculture were carried to create a tumor immunosuppressive microenvironment and to understand the role of DCLK1-isoform2 in such an environment. Organoids generated were counted, and wells were selected with an equal number of organoids. Selected wells with an equal number of organoids were seeded with M2-macrophages and T cells at a concentration of 1 × 105 cells per well of 48-well plates. After 24 hours, 1 nmol/L of commercially validated siRNA targeting human DCLK1 or 1 nmol/L human scrambled sequence (siSCR) was complexed with Lipofectamine 3000 and added to the wells. After 72 hours, organoids' coculture was used for cell viability and granzyme-B activity.

Cell viability assay

Viability of cells and spheroids using CellTiter-Glo 3D Cell Viability assay quantifying intratissue ATP content and this assay combines the enhanced penetration and lytic activity required for efficient lysis of three-dimensional (3D) cell culture with the generation of the stable ATP-dependent luminescent signal. Add a volume (100 μL) of CellTiter-Glo 3D Reagent equal to the volume of cell culture medium present in each well (1:1). Plates were vigorously mixed for 5 minutes using plate shaker and incubated for 25 minutes at room temperature. Record luminescence and results were represented in percentage viability.

Human cytokine array

The human cytokine array (Thermo Fisher Scientific) for 36 cytokine/chemokines was carried out according to the manufacturer's instructions. Briefly, the membrane was blocked using blocking buffer for an hour, and then the samples mixed with array antibodies were added to the membrane and incubated overnight at 4°C. Then the membranes were washed with wash buffer and developed using ECL, and images were captured and quantified.

Granzyme-B activity assay

The human granzyme-B enzyme hydrolyzes the specific substrate (Ac-IEPD-AFC) to release the quench of the fluorescent group, which can be detected fluorometrically at Ex/Em = 380/500 nm. The granzyme-B activity assay was carried out according to the manufacturer's instructions (Abcam). Briefly, the reaction mixture was added to each well containing cell lysates or positive controls or blank. Plates were incubated for 60 minutes at 37°C, and fluorescence was measured at Ex/Em = 380/500 nm.

Migration and invasion assay

For the invasion assay, matrigel-coated Transwells (BD Biosciences) were prepared by retrieving in serum-free media for 2 hours at 37°C. For the migration assay, Transwells (BD Biosciences) were used. Cell culture medium containing 10% FBS was added to the bottom of each well as a chemoattractant, and the cells were incubated in the transwell inserts for 24 hours at 37°C with 5% CO2. Afterward, a cotton swab was used to scrape noninvasive/migratory cells off the top of Transwells; the remaining cells were fixed and stained with 0.1% crystal violet and allowed to dry. After drying, all invading/migrating cells were counted from each Transwell. Results are reported as the percentage of cells invaded and/or migrated.

Quantitative real-time RT-PCR

Total RNA was isolated from a tumor or immune cells using Tri Reagent (MRC) as per the manufacturer's instructions. First-strand cDNA synthesis was carried out using SuperScript II Reverse Transcriptase and random hexanucleotide primers (Invitrogen). The complementary DNA was subsequently used to perform RT-PCR on an iCycler IQ5 Thermal Cycler (Bio-Rad) using SYBR Green (Molecular Probes) with gene-specific primers and JumpStart Taq DNA polymerase (Sigma). The crossing threshold value assessed was normalized to β-actin, and quantitative changes in mRNA were expressed as fold change relative to control ± SD value.

Immunoblot analysis

Twenty-five micrograms of the total protein were size-separated in a 4%–12% SDS polyacrylamide gel and transferred electrophoretically onto a PVDF membrane with a wet-blot transfer apparatus (Bio-Rad). The membrane was blocked and incubated overnight with a primary antibody and was subsequently incubated with horseradish peroxidase–conjugated secondary antibody. The proteins were detected using ECL Western blotting detection reagents (Amersham-Pharmacia). Protein density quantification was performed using GelQuant software. Actin (42 kD) was used as a loading control.

Immunohistochemistry/immunofluorescence

Standard IHC protocols were used with specific antibodies, as described previously (17, 27, 28). Immunohistochemistry: Heat-induced epitope retrieval was performed on 4-μm formalin-fixed, paraffin-embedded sections utilizing a pressurized Decloaking Chamber (Biocare Medical LLC) in citrate buffer (pH 6.0) at 99°C for 18 minutes. Brightfield: Slides were incubated in 3% hydrogen peroxide at room temperature for 10 minutes. After incubation with the primary antibody overnight at 4°C, slides were incubated in a Promark peroxidase-conjugated polymer detection system (Biocare Medical LLC) for 30 minutes at room temperature. After washing, slides were devolved with diaminobenzidine (Sigma-Aldrich). Fluorescence: Slides were incubated in normal serum and BSA blocking step at room temperature for 20 minutes. After incubation with primary antibody overnight at 4°C, slides were labeled with Alexa Fluor dye–conjugated secondary antibody and mounted with ProLong Gold (Invitrogen). Image Acquisition: Slides were examined on the Nikon Eclipse Ti motorized microscope paired with image app operated by the NIS-Elements Microscope Imaging Software platform (Nikon Instruments).

Statistical analyses

All statistical analyses were performed in GraphPad Prism 6.0, SPSS Statistics 22, and Microsoft Excel. One-way ANOVA and the Student t test were used to determine statistical significance. Pearson product-moment correlation was used for analysis and correlation of gene expressions between two groups. P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

Higher expression of DCLK1 in human pancreatic cancer predicts immunosuppressive phenotype

We previously reported that DLCK1 is highly expressed in human and mouse models of PDAC (13, 19). To address whether DCLK1-isoform2, an alternative splice variant of DCLK1, is highly expressed in patients with PDAC, we used TSVdb (22), an interactive web portal, to perform a comparative analysis on DCLK1 splicing variants from TCGA pancreatic cancer (PAAD) RNA-seq datasets. We found that DCLK1-isoform2 is highly expressed in pancreatic cancer, whereas DCLK1-isoform1 is marginally increased compared with solid tissue normal (Fig. 1A; Supplementary Fig. S2). We observed increased immunostaining for DCLK1-isoform2 in epithelial and stromal cells in human PDAC tissues (total 103 cases) compared with scant staining in normal adjacent tissues (Fig. 1B and C) using Abcam-31704 Ab which detects DCLK1-isoform2/4 (23). We analyzed the correlation between DCLK1 mRNA expression and immunosuppressive gene expressions using the TCGA PAAD RNA-seq database. The dataset revealed that increased DCLK1 was associated with increased expression of immunosuppressive markers (ARG1, ARG2, CCL17, CCL22, IL10, IL13, IL25, IL16, CCL1, CCL2, MIP, CXCL12, CD4, CD6, CTLA4, and ITGAM; Fig. 1D). Furthermore, a strong correlation was noted between DCLK1 mRNA and the M2-macrophages markers (CD163 and MRC1) compared with M1-macrophage markers (IL23A and IL1A; Fig. 1E). Collectively, our data for the first time indicate a potential link between DCLK1 and macrophage polarization toward an immunosuppressive M2 phenotype.

Figure 1.

DCLK1-isoform2 expression increased in PDAC and associates with immunosuppressive phenotype. A, DCLK1-isoform2 mRNA is highly expressed in pancreatic adenocarcinoma compared with adjacent solid pancreatic normal tissue in the TCGA PAAD dataset analyzed from TSVdb webtool. B, Human tissue microarrays (TMA) of pancreatic adenocarcinoma and adjacent normal tissues were stained for DCLK1-isoform2, and the intensity was scored. IHC reveals that DCLK1-isoform2 is highly expressed in pancreatic adenocarcinoma compared with normal adjacent pancreas issues. C, Representative graph of DCLK1 expression in normal and pancreatic adenocarcinoma TMA. DCLK1 mRNA and mRNA of immunosuppressive markers (ARG1, ARG2, CCL17, CCL22, IL10, IL13, IL25, IL16, CCL1, CCL2, MIP, CXCL12, CD4, CD6, CTLA4, and ITGAM; D) and macrophage markers (CD163, MRC1, IL23A, and IL1A; E) were downloaded from PAAD dataset of TCGA database. DCLK1 expression is positively correlated with genes of immunosuppressive markers and polarized macrophage M2 markers. Color indicates a correlation of DCLK1 and other genes: negative (green) and positive (red).

Figure 1.

DCLK1-isoform2 expression increased in PDAC and associates with immunosuppressive phenotype. A, DCLK1-isoform2 mRNA is highly expressed in pancreatic adenocarcinoma compared with adjacent solid pancreatic normal tissue in the TCGA PAAD dataset analyzed from TSVdb webtool. B, Human tissue microarrays (TMA) of pancreatic adenocarcinoma and adjacent normal tissues were stained for DCLK1-isoform2, and the intensity was scored. IHC reveals that DCLK1-isoform2 is highly expressed in pancreatic adenocarcinoma compared with normal adjacent pancreas issues. C, Representative graph of DCLK1 expression in normal and pancreatic adenocarcinoma TMA. DCLK1 mRNA and mRNA of immunosuppressive markers (ARG1, ARG2, CCL17, CCL22, IL10, IL13, IL25, IL16, CCL1, CCL2, MIP, CXCL12, CD4, CD6, CTLA4, and ITGAM; D) and macrophage markers (CD163, MRC1, IL23A, and IL1A; E) were downloaded from PAAD dataset of TCGA database. DCLK1 expression is positively correlated with genes of immunosuppressive markers and polarized macrophage M2 markers. Color indicates a correlation of DCLK1 and other genes: negative (green) and positive (red).

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Generation and viability of M1-macrophages

M1-macrophages were generated from THP1 cells and confirmed by both morphology and the expression of M1 markers (CD86 and HLA-DR; Fig. 2A and B). The viability of M1-macrophages was assessed by MTT assay at 0, 6, 12, 24, 48, 72, 96, and 120 hours. We observed a 5% to 8% reduction in the viability of M1-macrophages at 120 hours (Fig. 2C). Our data suggest that M1-macrophages derived from THP1 cells can survive up to 5 days without a significant loss in viability.

Figure 2.

Coculture of DCLK1-isoform2–overexpressing AsPC1 cells or MIA PaCA2 cells polarizes M1-macrophages into an M2 phenotype. A, Morphologic difference between THP1 cells and THP-1–derived macrophages (M1). B, Protein expression of M1-macrophage markers HLA-DR and CD86 between THP1 and M1-macrophages by immunoblot analysis. C, The line graph represents % macrophage viability up to 120 hours. D, Morphologic difference between AsPC1 vector control and AsPC1-DCLK1-isoform2–overexpressing cells. E, Protein expression of DCLK1-isoform2 in the AsPC1 vector control cells and AsPC1-DCLK1-isoform2–overexpressing cells by immunoblot analysis. F, mRNA expression of DCLK1-isoform2 in the AsPC1 vector control cells and AsPC1-DCLK1-isoform2–overexpressing cells by RT-PCR analysis. G, Protein expression of CD163 and CD206 (M2 markers) and CD86 and HLA-DR (M1 markers) in the total protein lysates of AsPC1 vector control cells+M1-macrophages treated with siScramble, AsPC1 vector control cells+M1-macrophages treated with siDCLK1, AsPC1-DCLK1-isoform2 cells+M1-macrophages treated with siScramble, and AsPC1-DCLK1-isoform2 cells+M1-macrophages treated with siDCLK1. H, Protein expression of CD163 and CD206 (M2 markers) and CD86 and HLA-DR (M1 markers) in the total protein lysates of MIA PaCa2 vector control cells+M1-macrophages treated with siScramble, MIA PaCa2 vector control cells+M1-macrophages treated with siDCLK1, MIA PaCa2-DCLK1-isoform2 cells+M1-macrophages treated with siScramble, and MIA PaCa2-DCLK1-isoform2 cells+M1-macrophages treated with siDCLK1. All quantitative data are expressed as mean ± SD of a minimum of three independent experiments. P values of <0.05 were considered statistically significant.

Figure 2.

Coculture of DCLK1-isoform2–overexpressing AsPC1 cells or MIA PaCA2 cells polarizes M1-macrophages into an M2 phenotype. A, Morphologic difference between THP1 cells and THP-1–derived macrophages (M1). B, Protein expression of M1-macrophage markers HLA-DR and CD86 between THP1 and M1-macrophages by immunoblot analysis. C, The line graph represents % macrophage viability up to 120 hours. D, Morphologic difference between AsPC1 vector control and AsPC1-DCLK1-isoform2–overexpressing cells. E, Protein expression of DCLK1-isoform2 in the AsPC1 vector control cells and AsPC1-DCLK1-isoform2–overexpressing cells by immunoblot analysis. F, mRNA expression of DCLK1-isoform2 in the AsPC1 vector control cells and AsPC1-DCLK1-isoform2–overexpressing cells by RT-PCR analysis. G, Protein expression of CD163 and CD206 (M2 markers) and CD86 and HLA-DR (M1 markers) in the total protein lysates of AsPC1 vector control cells+M1-macrophages treated with siScramble, AsPC1 vector control cells+M1-macrophages treated with siDCLK1, AsPC1-DCLK1-isoform2 cells+M1-macrophages treated with siScramble, and AsPC1-DCLK1-isoform2 cells+M1-macrophages treated with siDCLK1. H, Protein expression of CD163 and CD206 (M2 markers) and CD86 and HLA-DR (M1 markers) in the total protein lysates of MIA PaCa2 vector control cells+M1-macrophages treated with siScramble, MIA PaCa2 vector control cells+M1-macrophages treated with siDCLK1, MIA PaCa2-DCLK1-isoform2 cells+M1-macrophages treated with siScramble, and MIA PaCa2-DCLK1-isoform2 cells+M1-macrophages treated with siDCLK1. All quantitative data are expressed as mean ± SD of a minimum of three independent experiments. P values of <0.05 were considered statistically significant.

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DCLK1-isoform2–overexpressing AsPC1 cells polarize M1-macrophage into M2 phenotype

Our recent study reported increased expression of DCLK1-isoform2 and -isoform4 in renal cancer (23). The present data (Fig. 1A) indicate that only DCLK1-isoform2 is highly expressed in PDAC. To investigate its role in the polarization of M1-macrophages, DCLK1-isoform2 was overexpressed in AsPC1 cells (AsPC1-DCLK1-isoform2) and MIA PaCa2 cells (MIA PaCa2-DCLK1-isoform2). Measurement of protein and mRNA expression of DCLK1-isoform2 confirmed overexpression (Fig. 2E and F). AsPC1-DCLK1-isoform2 cells appear morphologically similar by 80% to 90% compared with the vector control cells, however, 10% to 20% displayed a spindle-like morphology (Fig. 2D). The coculture of AsPC1-DCLK1-isoform2 cells or MIA PaCa2-DCLK1-isoform2 cells with M1-macrophages resulted in a marked increase in expression of the M2-macrophage markers CD163 and CD206, and the reduction in the M1 markers, CD86 and HLA-DR (Fig. 2G and H). This increase in M2 marker expression was blocked when AsPC1-DCLK1-isoform2 cells or MIA PaCa2-DCLK1-isoform2 cells were treated with siRNA against DCLK1 before coculture (Fig. 2G and H). Taken together, these data strongly suggest that overexpression of DCLK1-isoform2 in the tumor cells promotes macrophage polarization into an M2 phenotype in vitro.

DCLK1-isoform2 promotes macrophage polarization via secreted cytokines/chemokines

To determine whether AsPC1-DCLK1-isoform2 cells require physical interaction or paracrine interaction to polarize macrophages, we performed dual-culture experiments where M1-macrophages and AsPC1-DCLK1-isoform2 cells were grown in transwells (Fig. 3A). We observed a distinct upregulation of M2 markers in the macrophages when dual-cultured with AsPC1-DCLK1-isoform2 cells, but not with vector control AsPC1 cells similar to that observed in coculture experiments (Fig. 3B), suggesting that secretory factors generated by AsPC1-DCLK1-isoform2 cells were responsible for the polarization. To identify the secreted cytokines/chemokines involved in this process, we performed cytokine protein profiling from the spent media of AsPC1-DCLK1-isoform2 cells and observed nine key chemokines/cytokines (CCL1, CCL2, MIP1a/b, CXL12, G-CSF, IL13, IL25, IL16, and IL21) that were greater than 2-fold compared with vector control cells (Fig. 3C and D). These key cytokines/chemokines have been reported previously to play an important role in macrophage polarization toward the M2 phenotype (29). Further studies are underway to specifically characterize the role of each soluble factor in DCLK1-dependent macrophage polarization.

Figure 3.

DCLK1-isoform2–overexpressing AsPC1 cells secrete cytokines/chemokines to polarize macrophages in a dual-culture system. A, Dual cell culture model. B, Protein expression of CD163 and CD206 (M2 markers) in the total protein lysates of M1-macrophages dual-cultured with AsPC1 vector control cells or M1-macrophages dual-cultured with AsPC1-DCLK1-isoform2 cells. C, Human cytokine array by dot blot analysis, conditioned media collected from AsPC1 vector control cell culture, or AsPC1-DCLK1-isoform2–overexpressing cell culture utilized for the array. Cytokines/chemokines with twofold differences in their increase were numbered in the blot and named. D, Arrays were quantified using the densitometric analysis to represent a fold change in a bar graph.

Figure 3.

DCLK1-isoform2–overexpressing AsPC1 cells secrete cytokines/chemokines to polarize macrophages in a dual-culture system. A, Dual cell culture model. B, Protein expression of CD163 and CD206 (M2 markers) in the total protein lysates of M1-macrophages dual-cultured with AsPC1 vector control cells or M1-macrophages dual-cultured with AsPC1-DCLK1-isoform2 cells. C, Human cytokine array by dot blot analysis, conditioned media collected from AsPC1 vector control cell culture, or AsPC1-DCLK1-isoform2–overexpressing cell culture utilized for the array. Cytokines/chemokines with twofold differences in their increase were numbered in the blot and named. D, Arrays were quantified using the densitometric analysis to represent a fold change in a bar graph.

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M2-macrophages enhance PDAC cell aggressiveness

To determine whether DCLK1-isoform2 polarized M2-macrophages contribute to tumor cell aggressiveness, we dual-cultured M2-macrophages or M1-macrophages with parental AsPC1 cells and evaluated the self-renewal and invasion/migration of tumor cells. We noted a marked increase in tumor cell invasion and migration (2–3-fold) when dual-cultured with DCLK1-isoform2–polarized M2-macrophages (Fig. 4A and B). We also observed an increased expression of epithelial–mesenchymal transition (EMT) factors SNAIL and SLUG in the PDAC cells (Fig. 4D). Self-renewal of parental AsPC1 cells increased 3-fold when dual-cultured with DCLK1-isoform2–induced M2-macrophages (Fig. 4F and G). Interestingly, DCLK1 silencing in parental AsPC1 cells reversed the M2-macrophage–mediated increase in SNAIL and SLUG, invasion, migration, and self-renewal (Fig. 4B, C, and E–G). To investigate the complex cytokine network between cancer cells and macrophages, we performed cytokine protein profiling from the spent media of M2-macrophages and observed key chemokines/cytokines [fold reduced: MIP1a/b (CCL3/4), CCL5, CXCL1, CXCL10, CXCL12, G-CSF, CD54, IL1b, IL8, IL13, IL18, IL21, IL32a, Serpin E1; fold increased: IL1ra, IL4, IL16] that were greater than 2-fold compared with M1-macrophages (Fig. 4H and I). These key cytokines/chemokines have been reported to play an important role in tumor aggression (30–32).

Figure 4.

DCLK1-isoform2–mediated M2-macrophages enhance PDAC aggression via soluble secretory factors. A, Migration and invasion of AsPC1 cells from M1-macrophage or M2-macrophage dual-culture. B, Bar graph represents % tumor cell migrated or invaded. C, Migration and invasion of AsPC1 cells either treated with siScramble or siDCLK1 from M2-macrophage dual-culture. D, Protein expression of SNAIL and SLUG in the AsPC1 cells dual cultured with M1- or M2-macrophages. E, Protein expression of SNAIL and SLUG in the AsPC1 cells treated with siScramble or siDCLK1 from a dual-culture experiment with M2-macrophages. F, Self-renewal of AsPC1 cells from M1-macrophage or M2-macrophage dual-culture; self-renewal of AsPC1 cells treated with siScramble, or siDCLK1 from M2-macrophage dual-culture. G, Bar graph represents the number of spheroids formed. H, Human cytokine array by dot blot analysis, conditioned media collected from M1- or M2-macrophages utilized for the array. Cytokines/chemokines with twofold differences in their increase or decrease were numbered in the blot and named. I, Arrays were quantified using the densitometric analysis to represent a fold change in a bar graph. All quantitative data are expressed as mean ± SD of a minimum of three independent experiments. P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

Figure 4.

DCLK1-isoform2–mediated M2-macrophages enhance PDAC aggression via soluble secretory factors. A, Migration and invasion of AsPC1 cells from M1-macrophage or M2-macrophage dual-culture. B, Bar graph represents % tumor cell migrated or invaded. C, Migration and invasion of AsPC1 cells either treated with siScramble or siDCLK1 from M2-macrophage dual-culture. D, Protein expression of SNAIL and SLUG in the AsPC1 cells dual cultured with M1- or M2-macrophages. E, Protein expression of SNAIL and SLUG in the AsPC1 cells treated with siScramble or siDCLK1 from a dual-culture experiment with M2-macrophages. F, Self-renewal of AsPC1 cells from M1-macrophage or M2-macrophage dual-culture; self-renewal of AsPC1 cells treated with siScramble, or siDCLK1 from M2-macrophage dual-culture. G, Bar graph represents the number of spheroids formed. H, Human cytokine array by dot blot analysis, conditioned media collected from M1- or M2-macrophages utilized for the array. Cytokines/chemokines with twofold differences in their increase or decrease were numbered in the blot and named. I, Arrays were quantified using the densitometric analysis to represent a fold change in a bar graph. All quantitative data are expressed as mean ± SD of a minimum of three independent experiments. P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

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Dclk1 and tumor immunosuppressive PDAC phenotype in vivo

We investigated the expression of Dclk1 and the relative abundance of the immunosuppressive cells of the pancreatic TME in KPCY (KRasLSLG12Dp53LSLR172HPdx1CreRosa26YFP) mice at 5 weeks (precancerous) and 20 weeks (PDAC) of age. We observed increased immunoreactive Dclk1-isoform2 in epithelial and stromal tissues of PDAC from 20-week-old KPCY mice compared with 5 weeks old (Fig. 5A; Supplementary Fig. S3). We observed a marked increase of the pan-myeloid marker (CD11B), which identifies myeloid, macrophage, and dendritic cells in 20-week-old KPCY tumors compared with 5 weeks old (Fig. 5A; Supplementary Fig. S3). We observed a distinct immunoreactive CD8 in 20-week-old KPCY mice tumor compared with 5 weeks old (Fig. 5B; Supplementary Fig. S3). However, within PDAC tumors, there is marked heterogeneity of T-cell infiltration. We obtained fixed CD8+ T-cell high (“T-cell high”) and CD8+ T-cell low (“T-cell low”) tumor tissues of autochthonous mouse model of pancreatic adenocarcinoma (20) as a generous gift from Ben Z. Stanger, MD, Ph.D., at the University of Pennsylvania. We performed IHC in these tissues and observed that the expression of DCLK1-isoform2 was inversely correlated with the infiltration of CD8+ T cells. DCLK1 staining was higher in CD8+ T-cell low tumor tissues and lower in CD8+ T-cell high tumor tissues (Fig. 5C; Supplementary Fig. S4). Following this observation, we observed a significant increase in the expression of arginase1 (M2 marker) in CD8+ T-cell low tumor tissues in contrast to CD8+ T-cell high tumor tissues (Fig. 5D; Supplementary Fig. S5). This suggests that the expression of DCLK1-isoform2 prevents the infiltration of CD8+ T cells through M2-macrophages.

Figure 5.

DCLK1-isoform2 expression positively and negatively correlates with M2-macrophages and CD8+ T cells, respectively, to induce immunosuppression in PDAC. A, IHC was performed to stain Dclk1 and CD11B in the pancreatic tissues collected from 5-week (precancerous) and 20-week (PDAC)-old KPCY mice (n = 5). B, CD8 staining was performed in the pancreatic tissues collected from 5-week- and 20-week-old KPCY mice. C, Fixed tumor tissues with CD8+ T-cell high (“T-cell high”) and CD8+ T-cell low (“T-cell low”) from an autochthonous mouse model (n = 5) of pancreatic adenocarcinoma were stained for Dclk1. D, Fixed tumor tissues with CD8+ T-cell high and CD8+ T-cell low from an autochthonous mouse model of pancreatic adenocarcinoma were stained for Dclk1 and Arginase1. E, Line graph represents the percentage proliferation of CD8+ T cells collected from M1-macrophage dual-culture and M2-macrophage dual-culture. F, Bar graph of relative fluorescence units (RFU) represents the granzyme-B activity of CD8+ T cells cocultured with AsPC1 or AsPC1+M1-macrophages or AsPC1+M2-macrophages. ***, compared with CD8+ T cells; ###, compared with M1+T cells+AsPC1 culture. All quantitative data are expressed as mean ± SD, and P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

Figure 5.

DCLK1-isoform2 expression positively and negatively correlates with M2-macrophages and CD8+ T cells, respectively, to induce immunosuppression in PDAC. A, IHC was performed to stain Dclk1 and CD11B in the pancreatic tissues collected from 5-week (precancerous) and 20-week (PDAC)-old KPCY mice (n = 5). B, CD8 staining was performed in the pancreatic tissues collected from 5-week- and 20-week-old KPCY mice. C, Fixed tumor tissues with CD8+ T-cell high (“T-cell high”) and CD8+ T-cell low (“T-cell low”) from an autochthonous mouse model (n = 5) of pancreatic adenocarcinoma were stained for Dclk1. D, Fixed tumor tissues with CD8+ T-cell high and CD8+ T-cell low from an autochthonous mouse model of pancreatic adenocarcinoma were stained for Dclk1 and Arginase1. E, Line graph represents the percentage proliferation of CD8+ T cells collected from M1-macrophage dual-culture and M2-macrophage dual-culture. F, Bar graph of relative fluorescence units (RFU) represents the granzyme-B activity of CD8+ T cells cocultured with AsPC1 or AsPC1+M1-macrophages or AsPC1+M2-macrophages. ***, compared with CD8+ T cells; ###, compared with M1+T cells+AsPC1 culture. All quantitative data are expressed as mean ± SD, and P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

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M2-macrophages inhibit CD8+ T-cell activity

We investigate whether DCLK1-isoform2–polarized M2-macrophages can inhibit CD8+ T-cell proliferation and the cytotoxic activity in PDAC. CD8+ T cells when dual-cultured with tumor-educated M2-macrophages exhibited a 25% reduction in its proliferation compared with M1-macrophage dual culture (Fig. 5F). The activity of granzyme-B was significantly reduced (2.5-fold) in CD8+ T cells when cocultured with AsPC1 cells+M2-macrophages compared with CD8+ T cells cocultured with AsPC1 cells+M1-macrophages (Fig. 5G). Taken together, DCLK1-isoform2–expressing tumor cells induced M2-macrophages inhibit proliferation and cytotoxic function of CD8+ T cells.

Tumor organoid coculture model as immunosuppressive TME and role of DCLK1

To further understand the relevant immunomodulatory role of DCLK1-isoform2 in PDAC TME, we used a 3D coculture model that contains tumor organoids, CD8+ T cells, and M2 macrophages. Three-dimensional organoid models are relevant models to test for therapeutic strategies in a cocultures system (33, 34). Immune cells including M2-macrophages and CD8+ T cells were cocultured with tumor organoids in a 3D environment which recapitulates the tumor microenvironment and its complex immune and cellular interactions (Fig. 6A). After 24 hours of the addition of immune cells into the organoids, 3D cultures were treated with siScramble or siDCLK1, and the cultures were monitored for 72 hours. We observed a significant reduction in the number of organoids in siDCLK1-treated 3D cultures compared with siScramble treatment (Fig. 6B and C). Compared with 0 hour, siScramble treatment enhanced the organoid numbers at 72 hours, which suggests that M2-macrophages are immunosuppressive and enhance tumor aggressiveness. On the other hand, inhibition of DCLK1 enhanced the cytotoxic activity of CD8+ T cells by increasing the granzyme-B activity (Fig. 6D) and reduced the viability of 3D cultures (Fig. 6E). Our 3D TME data enable a better understanding of the immunomodulatory profile of DCLK1-isoform2 and can be used to test DCLK1-related drug testing or as adjuvant therapy with other immunomodulatory therapies.

Figure 6.

Inhibition of DCLK1-isoform2 in the PDAC organoid coculture system enhanced the cytotoxic function of CD8+ T cells and reduced viability of tumor organoid. A, Organoids generated from MIA PaCa2 cells and organoids were cocultured with M2-macrophages and CD8+ T cells. B, Organoid cocultures were treated with siScramble or siDCLK1 and monitored up to 72 hours. C, siDCLK1 reduced the number of organoids compared with siScramble treatment to the organoid coculture system, and the bar graph represents the number of organoids. D, Bar graph of relative fluorescence units (RFU) represents the granzyme-B activity of CD8+ T cells cocultured with organoids+M2-macrophages treated with siScramble or siDCLK1. E, Bar graph of percentage cell viability between organoid cocultures treated with siScramble or siDCLK1 at 0 and 72 hours. All quantitative data are expressed as mean ± SD, and P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

Figure 6.

Inhibition of DCLK1-isoform2 in the PDAC organoid coculture system enhanced the cytotoxic function of CD8+ T cells and reduced viability of tumor organoid. A, Organoids generated from MIA PaCa2 cells and organoids were cocultured with M2-macrophages and CD8+ T cells. B, Organoid cocultures were treated with siScramble or siDCLK1 and monitored up to 72 hours. C, siDCLK1 reduced the number of organoids compared with siScramble treatment to the organoid coculture system, and the bar graph represents the number of organoids. D, Bar graph of relative fluorescence units (RFU) represents the granzyme-B activity of CD8+ T cells cocultured with organoids+M2-macrophages treated with siScramble or siDCLK1. E, Bar graph of percentage cell viability between organoid cocultures treated with siScramble or siDCLK1 at 0 and 72 hours. All quantitative data are expressed as mean ± SD, and P values of <0.05 (*), <0.01 (**), and 0.001 (***) were considered statistically significant.

Close modal

There is a large body of evidence that the TME has a tremendous role in regulating tumor-mediated immunosuppression. This complex interaction between the tumor and several immune cell types creates an enormous challenge for the development of novel therapeutic agents to treat PDAC. In this study, we describe a new role for DCLK1-isoform2 in promoting macrophage polarization toward the immunosuppressive phenotype via a paracrine-dependent mechanism. These M2-macrophages advance cancer cell invasion, migration, and self-renewal and reduce cytotoxic T-cell function. Our studies illuminate the mechanistic interaction of M2-macrophages and the tumor epithelium. Interestingly, we demonstrate that these interactions are mediated via DCLK1-isoform2–dependent mechanism.

Macrophages are an incredibly diverse set of immune cells that have been hijacked by tumor cells to favor tumor progression and induce immunosuppression (29). However, the mechanism by which tumor cells polarize macrophages toward an M2 phenotype has not yet been fully explored. Indeed, in PDAC, tumor-associated macrophages (TAM) are the most abundant immune cell population in the TME (4, 35). We used coculture and dual-culture system to evaluate the role of DCLK1-isoform2 in macrophage polarization. We reveal that overexpression of DCLK1-isoform2 in PDAC cells plays a major role in macrophage polarization by enhancing the secretion of key cytokines/chemokines that have been associated with the macrophage polarization process (29). Silencing DCLK1 inhibits this process and influences macrophages to retain the M1 phenotype. Interestingly, our studies reveal the findings that these DCLK1-isoform2–polarized M2-macrophages possess the ability to increase the aggressiveness (invasion/migration) and self-renewal capacity of the parental pancreatic cancer cell line. DCLK1 silencing in the parental pancreatic cancer cell line albeit low at baseline completely abrogated M2-macrophage ability to enhance the aggressiveness and self-renewal of PDAC cells. These data suggest that M2-macrophages are a major component of pancreatic cancers and can promote tumorigenesis by facilitating invasion, migration, and self-renewal via key secretory cytokines (31, 36). Our data also suggest that interaction between DCLK1-isoform2–expressing epithelial cells and M1-macrophages creates a feedback loop that involves signaling from the tumor cells to M1-macrophages and communication between M2-macrophages and epithelial cells that require DCLK1-isoform2. The studies presented here demonstrate that in mouse pancreatic tumor tissues, elevated Dclk1-isoform2 expression is associated with high levels of M2-macrophages and low levels of CD8+ T-cell infiltration. In fact, there is an inverse correlation observed between Dclk1-isoform2 or M2-macrophages and T-cell infiltration. One main function of the M2-macrophage is to induce immunosuppressive phenotype by inhibiting the cytotoxic activity of CD8+T cells and induce senescence (5, 8, 37). We report here the immunosuppressive role of DCLK1-isoform2–overexpressing PDAC cell–educated M2-macrophages which reduced the proliferation and cytotoxic activity of CD8+ T cells. However, further molecular studies are required to identify the key factors from M2-macrophages involved in the deactivation of CD8+ T cells. Furthermore, to understand the immunomodulatory role of DCLK1-isoform2 in the PDAC TME, we developed 3D cocultures of tumor organoids and immune cells and inhibited DCLK1 by siRNA treatment. We demonstrated that our 3D coculture model is relevant in vitro tool for testing human immune therapies including DCLK1 therapy. We demonstrated that inhibition of DCLK1-isoform2 in the 3D coculture enhanced the cytotoxic function of CD8+ T cells and thus reduced the number of organoids.

Our studies link DCLK1-isoform2 expression in tumor cells to the regulation of a major component of the innate immune system, the M2-macrophage, which is a predominant immune cell type in PDAC. Our findings thus signify the potential role of DCLK1-isoform2 in tumor cell–mediated macrophage polarization and associated immunosuppression for tumor aggressiveness. Although future studies are required to definitively link DCLK1-isoform2 and protumorigenic features, targeting DCLK1-isoform2 in PDAC may represent a novel therapeutic approach to reactivating host antitumor immunity.

C.W. Houchen has an ownership interest (including patents) in COARE Holdings Inc. No potential conflicts of interest were disclosed by the other authors.

All authors are aware of the content and agree with the submission.

Conception and design: P. Chandrakesan, J. Panneerselvam, C.V. Rao, C.W. Houchen

Development of methodology: P. Chandrakesan, J. Panneerselvam, D. Qu, C.W. Houchen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Chandrakesan, J. Panneerselvam, R. May, D. Qu, K. Pitts, C.W. Houchen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Chandrakesan, N. Weygant, D. Qu, C.W. Houchen

Writing, review, and/or revision of the manuscript: P. Chandrakesan, J. Panneerselvam, N. Weygant, D. Qu, K. Pitts, B.Z. Stanger, C.V. Rao, M.S. Bronze, C.W. Houchen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Chandrakesan, W.R. Berry, C.V. Rao, C.W. Houchen

Study supervision: P. Chandrakesan, C.W. Houchen

Others (provided reagents): B.Z. Stanger

This work was supported by DOD grant no. W81XWH-18-1-0457 (principal investigator: P. Chandrakesan) and NIH grant no. 1R01CA182869-01A1 (principal investigator: C.W. Houchen). We thank Dr. Stanley Lightfoot, OUHSC, Oklahoma, for the histology and pathology evaluation. We thank Landon Moore and Naushad Ali, OUHSC, Oklahoma, for language edit.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data