Macrophages In Pancreatic Ductal Adenocarcinoma Research Articles

Ectopic CXCR2 expression cells improve the anti-tumor efficiency of CAR-T cells and remodel the immune microenvironment of pancreatic ductal adenocarcinoma

BackgroundRecent progressions in CAR-T cell therapy against pancreatic ductal adenocarcinoma (PDAC) remain disappointing, which are partially attributed to the immunosuppressive microenvironment including macrophage-mediated T cell repletion.MethodsWe first characterized the expression patterns of macrophage-relevant chemokines and identified CXCR2 as the key factor regulating T cell trafficking and tumor-specific accumulation in PDAC microenvironment. After that, we synthesized and introduced a CXCR2 expression cascade into Claudin18.2 CAR-T cells and compared the behaviors of CAR-T cells in vitro and in vivo. The therapeutic potential of CXCR2 CAR-T was evaluated in two different allogeneic models: subcutaneous allografts and metastatic PDAC models.ResultsThe results showed that CXCR2 CAR-T not only reduced the size of allografted PDAC tumors, but also completely eliminated the formation of metastases. Lastly, we investigated the tumor tissues and found that expression of ectopic CXCR2 significantly improved tumor-targeted infiltration and residence of T cells and reduced the presence of MDSCs and CXCR2 + macrophages in PDAC microenvironment.ConclusionOur studies suggested that ectopic CXCR2 played a significant and promising role in improving the efficiency of CAR-T therapy against primary and metastatic PDAC and partially reversed the immune-suppressive microenvironment.

Abstract B041: Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma

Abstract Background. Keratin 17 (K17) is a negative prognostic and predictive biomarker, overexpressed in the biologically most aggressive forms of pancreatic ductal adenocarcinoma (PDAC). In other anatomic sites and disease processes, K17 expression impacts the inflammatory microenvironment, correlating with changes in the expression of inflammatory cytokines and the infiltration of CD8+ T cells. Thus, we hypothesized that K17 expression also impacts the immune response in PDAC. Methods. We aimed to determine the relationship between intratumoral and peritumoral immune cell infiltrates versus K17 expression in tumor cells, using brightfield multiplexed immunohistochemistry (mIHC) and our suite of deep learning tools to quantitatively evaluate the expression of four biomarkers of T-cells and macrophages. The study included a multi-institutional cohort of 235 PDAC cases from Stony Brook University Hospital (n=54), Thomas Jefferson University (n=67), Cedars Sinai Medical Center (n=7) and Know Your Tumor PanCAN-Perthera (n=107). Peritumoral immune cells were scored in zones defined as stromal tissue within ≤25 µm of the closest tumor cell. Intratumor immune cells were defined as those that physically touched or overlapped with tumor cells. Immune cell counts in peritumoral and intratumoral K17-negatvie areas of the tumor were normalized to immune cell counts in K17-positive areas. Results. The density of CD8+ T cells in K17-negative peritumoral zones was greater than in K17-positive peritumoral zones in 82.6% of PDACs. Similarly, the density of intratumoral CD8+ T cells in K17-negative areas was greater than in K17-positive areas in 93.2% of cases. Less striking inverse correlations for immune cell density relative to K17 expression were found for CD4+ peritumor lymphocytes and both intratumoral and peritumor CD16+ macrophages. By contrast, CD163+ (tumor-promoting) macrophages were more numerous in K17+ peritumoral and intratumoral zones. All differences were statistically significant and independent of tumor grade, stage, histologic subtype, and neoadjuvant treatment history (p< 0.0001). Conclusions. Correlations between K17 and inflammatory cells suggests K17 expression negatively influences the number and infiltration of T-cells and contributes to the marked immunosuppressive effect of PDAC. These findings suggest that the biologic impact of K17 goes beyond its role as a negative prognostic biomarker that impacts tumor growth, metastasis, and chemoresistance, to suppress the immune response to PDAC. Understanding how K17 influences immune cell function may inform future immunotherapeutic opportunities for PDAC treatment. Citation Format: Lyanne Oblein, Sruthi Babu, Mariana Torrente-Goncalves, Lucia Roa, Michael Horowitz, Mahmudul Hasan, Danielle Fassler, Jaymie Oentoro, Emanuel Petricoin, Lynn M. Matrisian, Natalia Marchenko, Edik Blais, Felicia Allard, Ali Alkalin, Wei Jiang, Brent Larson, Andrew Hendifar, Chao Chen, Shahira Abousamra, Dimitris Samaras, Tahsin Kurc, Joel Saltz, Luisa Escobar-Hoyos, Kenneth Shroyer. Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B041.

AMIGO2 is a pivotal therapeutic target related to M2 polarization of macrophages in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a common kind of lethal cancer, with low early diagnostic rate and poor prognosis. In this study, we identified and verified the AMIGO2 with significant diagnostic and prognostic value in PDAC through LASSO regression combined with multiple machines learning methods, including RVM-RFE and Random Forest in TCGA and GEO datasets. The relevance between the expression of AMIGO2 and M2 polarization of macrophages was identified through pancancer, normal tissue, and cell lines data in TCGA, GTEx and CCLE datasets. The relevance between AMIGO2 and M2 polarization was then further identified in our local PDAC cohort. Finally, the role of AMIGO2 as cancer promoter and pivotal factor enrolled in M2 polarization was verified through siRNA transfection and M2 macrophages induction. These findings could facilitate diagnosis and treatment of PDAC. In addition, further research was deemed necessary on the deep mechanism between AMIGO2 and M2 polarization of macrophages in PDAC.

Multi-omics analysis reveals the chemoresistance mechanism of proliferating tissue-resident macrophages in PDAC via metabolic adaptation

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically demonstrates resistance to chemotherapy. Tumor-associated macrophages (TAMs) are essential in tumor microenvironment (TME) regulation, including promoting chemoresistance. However, the specific TAM subset and mechanisms behind this promotion remain unclear. We employ multi-omics strategies, including single-cell RNA sequencing (scRNA-seq), transcriptomics, multicolor immunohistochemistry (mIHC), flow cytometry, and metabolomics, to analyze chemotherapy-treated samples from both humans and mice. We identify four major TAM subsets within PDAC, among which proliferating resident macrophages (proliferating rMφs) are strongly associated with poor clinical outcomes. These macrophages are able to survive chemotherapy by producing more deoxycytidine (dC) and fewer dC kinases (dCKs) to decrease the absorption of gemcitabine. Moreover, proliferating rMφs promote fibrosis and immunosuppression in PDAC. Eliminating them in the transgenic mouse model alleviates fibrosis and immunosuppression, thereby re-sensitizing PDAC to chemotherapy. Consequently, targeting proliferating rMφs may become a potential treatment strategy for PDAC to enhance chemotherapy.

Abstract C071: Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma

Abstract Background: Keratin 17 (K17) is a negative prognostic biomarker, overexpressed in the biologically most aggressive forms of pancreatic ductal adenocarcinoma (PDAC). In other anatomic sites and disease processes, K17 expression also correlates with immune cell infiltrates and could block T-cell infiltration. Thus, we hypothesized that K17 expression correlates with the inflammatory microenvironment in PDAC. In this study, we aimed to determine the relationship between the stromal immune cell infiltrates and K17 expression, using multiplexed immunohistochemistry (mIHC) and our suite of deep learning tools to quantitatively evaluate the expression of four biomarkers of T-cells and macrophages in PDAC. Methods: mIHC was performed on representative sections of 201 primary PDACs from Stony Brook University Hospital, Thomas Jefferson University Hospital, Cedars Sinai Medical Center, and from a national cohort (KYT, Pancreatic Cancer Action Network, and Perthera). Antibodies for CD4 (helper T-cells), CD8 (cytotoxic T-cells), CD16 (pan-macrophage), CD163 (M2 macrophages), pancytokeratin, and K17 were provided by Roche Diagnostics Corporation through a sponsored research agreement. mIHC was performed on a Discovery Ultra Autostainer (Roche), using horseradish peroxidase (HRP) and alkaline phosphatase (AP)-based protocols with multiple chromogens (Red: CD4, Purple: CD8, Yellow: CD16, Green: CD163, Teal: pancytokeratin, and Brown: K17) to enable multispectral imaging of diverse immune cell populations within the cancer microenvironment. A deep learning analysis workflow was used to detect and classify stromal inflammatory cells, in whole slide images (WSIs), generated using an Olympus VS120 digital microscope (Olympus, Tokyo, Japan). Pixel-wise predictions from a color auto-encoder (ColorAE) union UNET anchor UNET model were combined to create multi-class masks that were further analyzed to perform detection and classification. Results: The analysis of the inflammatory microenvironment focused on defining immune cell infiltrates located within 25 microns of the closest K17-positive versus K17-negative tumor cell in each representative section. Across the sum of K17-positive and negative zones/section, CD4 cell counts ranged from 0-10,617 (mean 2,709), CD8 cell counts ranged from 63-28,596 (mean 6,745), CD16 cell counts ranged from 4-7,797 (mean 3,024), and CD163 cell counts ranged from 35-34,696 (mean 14,968). CD4 T-helper cells, CD8 cytotoxic T cells, and CD16 macrophages were more numerous (respectively, p=0.0012; p=<0.0001; p=<0.0001) in K17-negative tumor zones compared to K17-positive zones. By contrast, the number of CD163 (M2) tumor-promoting macrophages was greater in K17 positive zones (p=0.0019). Conclusion: K17 expression by tumor cells impacts the chronic inflammatory microenvironment, shielding tumor cells from immune cell mediated cytotoxic responses, while recruiting tumor-promoting M2 macrophages, indicating that K17 impacts the immune response as a fundamental hallmark of aggression in PDAC. Citation Format: Lyanne Oblein, Michael Horowitz, Mahmudul Hasan, Sruthi Babu, Mariana Torrente-Goncalves, Lucia Roa, Jaymie Oentoro, Jason Harper, Xin Yao Zheng, Wei Jiang, Andrew Hendifar, Natalie Moshayedi, Brent Larson, Veronica Placencio-Hickok, Edik Blais, Emmanuel Petricoin, Joel Saltz, Natalia D. Marchenko, Luisa F. Escobar-Hoyos, Kenneth Shroyer. Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C071.

Single Cell RNA-Seq Identifies Immune-Related Prognostic Model and Key Signature-SPP1 in Pancreatic Ductal Adenocarcinoma.

There are no reliable biomarkers for early diagnosis or prognosis evaluation in pancreatic ductal adenocarcinoma (PDAC). Multiple scRNA-seq datasets for PDAC were retrieved from online databases and combined with scRNA-seq results from our previous study. The malignant ductal cells were identified through calculating copy number variation (CNV) scores. The robust markers of malignant ductal cells in PDAC were found. Five immune-related signatures, including SPP1, LINC00683, SNHG10, LINC00237, and CASC19, were used to develop a risk score formula to predict the overall survival of PDAC patients. We also constructed an easy-to-use nomogram, combining risk score, N stage, and margin status. The expression level of SPP1 was related to the prognosis and immune regulators. We found that SPP1 was mainly expressed in ductal cells and macrophages in PDAC. In conclusion, we constructed a promising prognostic model based on immune-related signatures for PDAC using scRNA-seq and TCGA_PAAD datasets.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ Tcells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

The CTCF/LncRNA-PACERR complex recruits E1A binding protein p300 to induce pro-tumour macrophages in pancreatic ductal adenocarcinoma via directly regulating PTGS2 expression.

BackgroundTumour‐associated macrophages (TAMs) play an important role in promoting the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to study the epigenetic mechanisms in regulating pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.MethodsThis study was conducted based on ex vivo TAMs isolated from PDAC tissues and in vitro THP1‐derived TAM model. RNA‐sequencing (RNA‐seq), assay for transposase‐accessible chromatin with sequencing and chromatin immunoprecipitation sequencing were performed to investigate gene expression, chromatin accessibility, transcription factor binding sites and histone modifications. Gene knockdown in THP1‐derived TAMs was performed with lentivirus, and the impact of THP1‐derived TAMs on invasion and metastasis ability of PDAC cells were investigated with in vitro and in vivo functional assays. RNA‐chromatin interaction was analysed by chromatin isolation through RNA purification with sequencing. RNA‐protein interaction was studied by RNA immunoprecipitation and RNA pull‐down.ResultsOur data showed that the transcription factor CTCF (CCCTC‐binding factor) was highly expressed in TAMs and predicted to be significantly enriched in hyper‐accessible chromatin regions when compared to monocytes. High infiltration of CTCF+ TAMs was significantly associated with poor prognosis in PDAC patients. Knockdown of CTCF in THP1‐derived TAMs led to the down‐regulation of specific markers for M2‐polarised TAMs, including CD206 and CD163. When THP1‐derived TAMs with CTCF knockdown, they showed a decreased ability of invasion and metastasis. Further integrative analysis of multi‐omics data revealed that prostaglandin‐endoperoxide synthase 2 (PTGS2) and PTGS2 antisense NF‐κB1 complex‐mediated expression regulator RNA (PACERR) were critical downstream targets of CTCF and positively correlated with each other, which are closely situated on a chromosome. Knockdown of PACERR exhibited a similar phenotype as observed in CTCF knockdown THP1‐derived TAMs. Moreover, PACERR could directly bind to CTCF and recruit histone acetyltransferase E1A binding protein p300 to the promoter regions of PACERR and PTGS2, thereby enhancing histone acetylation and gene transcription, promoting the M2 polarization of TAMs in PDAC.ConclusionsOur study demonstrated a novel epigenetic regulation mechanism of promoting pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.

Tumor-Associated Macrophages in Pancreatic Ductal Adenocarcinoma: Therapeutic Opportunities and Clinical Challenges.

Simple SummaryMacrophages are a major component of the pancreatic tumor microenvironment, and their increased abundance is associated with poor patient survival. Given the multi-faceted role of macrophages in promoting pancreatic tumor development and progression, these cells represent promising targets for anti-cancer therapy.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a 5-year survival rate of less than 10%. Macrophages are one of the earliest infiltrating cells in the pancreatic tumor microenvironment, and are associated with an increased risk of disease progression, recurrence, metastasis, and shorter overall survival. Pre-clinical studies have demonstrated an unequivocal role of macrophages in PDAC by contributing to chronic inflammation, cancer cell stemness, desmoplasia, immune suppression, angiogenesis, invasion, metastasis, and drug resistance. Several macrophage-targeting therapies have also been investigated in pre-clinical models, and include macrophage depletion, inhibiting macrophage recruitment, and macrophage reprogramming. However, the effectiveness of these drugs in pre-clinical models has not always translated into clinical trials. In this review, we discuss the molecular mechanisms that underpin macrophage heterogeneity within the pancreatic tumor microenvironment, and examine the contribution of macrophages at various stages of PDAC progression. We also provide a comprehensive update of macrophage-targeting therapies that are currently undergoing clinical evaluation, and discuss clinical challenges associated with these treatment modalities in human PDAC patients.

Tumor-Associated Macrophages in Pancreatic Ductal Adenocarcinoma: Origin, Polarization, Function, and Reprogramming.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy. PDAC is only cured by surgical resection in its early stage, but there remains a relatively high possibility of recurrence. The development of PDAC is closely associated with the tumor microenvironment. Tumor-associated macrophages (TAMs) are one of the most abundant immune cell populations in the pancreatic tumor stroma. TAMs are inclined to M2 deviation in the tumor microenvironment, which promotes and supports tumor behaviors, including tumorigenesis, immune escape, metastasis, and chemotherapeutic resistance. Herein, we comprehensively reviewed the latest researches on the origin, polarization, functions, and reprogramming of TAMs in PDAC.

IGFBP2 promotes tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma through the STAT3 pathway.

Tumor-associated macrophages (TAMs) represent the M2-like phenotype with potent immunosuppressive activity, and play a pro-tumor role in pancreatic ductal adenocarcinoma (PDAC) biology. In this study, we investigated the role of the insulin-like growth factor binding protein 2 (IGFBP2) as a determinant of TAM polarity. Clinical data revealed that the levels of IGFBP2 correlated with M2 TAMs accumulation and disease progression in human PDAC. In vivo mouse model experiments showed that IGFBP2 promoted an immunosuppressive microenvironment and tumor growth in a macrophage dependent manner. Bioinformatics analysis of PDAC transcriptomes revealed a significant association between IGFBP2 expression and M2 macrophage polarization and signal transducer and activator of transcription 3 (STAT3) activation. Mechanistic investigations demonstrated that IGFBP2 augmented the expression and secretion of IL-10 through STAT3 activation in PDAC cells, which induced TAM polarization toward an M2 phenotype. IGFBP2-polarized M2 macrophages significantly increased Tregs infiltration and impaired antitumor T-cell immunity in a mouse model. Thus, our investigations have illuminated the IGFBP2 signaling pathway that contributes to the macrophage-based immunosuppressive microenvironment in PDAC, suggesting that blocking the IGFBP2 axis constitutes a potential treatment strategy to reset TAM polarization toward an antitumor state in PDAC.

Oxidative stress induces monocyte-to-myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma.

BackgroundCancer‐associated fibroblasts (CAFs) are among the most prominent cells during the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC). However, CAFs are heterogeneous and the precise origins are not fully elucidated. This study aimed to explore whether monocytes can transdifferentiate into fibroblasts in PDAC and evaluate the clinical significance of this event.MethodsCD14+ monocytes were freshly isolated from human peripheral blood. Immunofluorescence, reverse transcription‐quantitative PCR, western blot, flow cytometry and enzyme‐linked immunosorbent assay were used to detect the expression of αSMA, fibronectin, and other relevant molecules. In addition, latex beads with a mean particle size of 2.0 µm were used to assess the phagocytic capacity. Moreover, RNA sequencing (RNA‐seq) was performed to identify the differences induced by H2O2 and the underlying mechanisms.ResultsImmunofluorescence identified αSMA and fibroblast‐specific protein 1 expression by tumor‐associated macrophages in PDAC. The in vitro experiment revealed that oxidative stress (H2O2 or radiation) induced monocyte‐to‐myofibroblast transdifferentiation (MMT), as identified by upregulated αSMA expression at both the RNA and protein levels. In addition, compared with freshly isolated monocytes, human monocyte‐derived macrophages increased fibronectin expression. RNA‐seq analysis identified p53 activation and other signatures accompanying this transdifferentiation; however, the p53 stabilizer nutlin‐3 induced αSMA expression through reactive oxygen species generation but not through the p53 transcription/mitochondria‐dependent pathway, whereas the p38 inhibitor SB203580 could partially inhibit αSMA expression. Finally, MMT produced a unique subset of CAFs with reduced phagocytic capacity that could promote the proliferation of pancreatic cancer cells.ConclusionsOxidative stress in the tumor microenvironment could induce MMT in PDAC, thus inducing reactive stroma, modulating immunosuppression, and promoting tumor progression. Reducing oxidative stress may be a promising future therapeutic regimen.

Pancreatic stromal Gremlin 1 expression during pancreatic tumorigenesis

Chronic pancreatitis (CP) is a major risk factor of pancreatic ductal adenocarcinoma (PDAC). How CP promotes pancreatic oncogenesis remains unclear. A characteristic feature of PDAC is its prominent desmoplasia in the tumor microenvironment, composed of activated fibroblasts and macrophages. Macrophages can be characterized as M1 or M2, with tumor-inhibiting or -promoting functions, respectively. We reported that Gremlin 1 (GREM1), a key pro-fibrogenic factor, is upregulated in the stroma of CP. The current study aimed to investigate the expression of GREM1 and correlation between GREM1 and macrophages within the pancreas during chronic inflammation and the development of PDAC. By mRNA in situ hybridization, we detected GREM1 mRNA expression within α-smooth muscle actin (SMA)-positive fibroblasts of the pancreatic stroma. These designated FibroblastsGrem1+ marginally increased from CP to pancreatic intraepithelial neoplasia (PanIN) and PDAC. Within PDAC, FibroblastsGrem1+ increased with higher pathological tumor stages and in a majority of PDAC subtypes screened. Additionally, FibroblastsGrem1+ positively correlated with total macrophages (MacCD68+) and M2 macrophages (M2CD163+) in PDAC. To begin exploring potential molecular links between FibroblastsGrem1+ and macrophages in PDAC, we examined the expression of macrophage migration inhibitory factor (MIF), an endogenous counteracting molecule of GREM1 and an M1 macrophage promoting factor. By IHC staining of MIF, we found MIF to be expressed by tumor cells, positively correlated with GREM1; by IHC co-staining, we found MIF to be negatively correlated with M2CD163+ expression. Our findings suggest that GREM1 expression by activated fibroblasts may promote PDAC development, and GREM1/MIF may play an important role in macrophage phenotype.

Induced CD10 expression during monocyte-to-macrophage differentiation identifies a unique subset of macrophages in pancreatic ductal adenocarcinoma

ObjectiveTumor associated macrophages (TAMs) promoted pancreatic ductal adenocarcinoma (PDAC) initiation and progression. In this study we aimed to evaluate CD10 expression by monocytes/macrophages and its clinical significance in PDAC. MethodsHuman CD14+ peripheral blood monocytes were isolated and cultured for 6–7 days to differentiate into macrophages in vitro. Monocytic THP-1 cells were cultured and treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 72 h to induce macrophage differentiation. Reverse transcription-quantitative PCR, immunohistochemistry, immunofluorescence, multiplex immunohistochemical staining and flow cytometry were performed to detect CD10 expression. In addition, the correlations between CD10 expression and immune cells infiltration were investigated through TIMER or GEPIA. Finally, Kaplan-Meier plotter and GEPIA databases were adopted to evaluate the influence of CD10 on clinical prognosis. ResultsOur results indicated that CD10 was expressed by a subset of human monocytes and many more cells expressed CD10 after differentiation into macrophages in vitro (13.19% vs. 41.39%; P < 0.0001). As for PDAC tissues, CD10 was correlated with immune cells infiltration and was expressed by a subset of TAMs. For THP-1 cells, PMA could induce CD10 expression through the MAPK pathway. The Kaplan-Meier plotter results suggested that CD10 expression had an impact on the prognosis of PDAC. ConclusionsIn this study we demonstrated that CD10 was expressed by human primary monocytes, human monocyte-derived macrophages and TAMs, and was correlated with poor prognosis in PDAC, suggesting CD10 to be a potential therapeutic target in PDAC.

Abstract 4517: Tumor-associated macrophages are reprogrammed by pancreatic ductal adenocarcinoma cells through tumor-induced DNA methylation on metabolic genes

Abstract Metabolism is shifted toward glycolysis with increased glucose uptake in anti-tumor M1 macrophages, and toward oxidative phosphorylation with significant decreased glucose uptake in pro-tumor M2 macrophages in Pancreatic ductal adenocarcinoma (PDA). Current study aims to investigate the mechanism by which tumor cells reprogram tumor associated macrophages metabolically and functionally. Using mouse bone marrow derived macrophages and human macrophages derived from peripheral blood mononuclear cells for contact and non-contact co-culture experiments with PDA tumor cells, we showed PDA tumor cells, through direct cell-cell contact confirmed by Lucifer yellow dye-coupling assay, induced DNA methylation and downregulation of a panel of glucose metabolism and oxidative phosphorylation genes selectively in M1 but not M2 macrophages by methylation specific PCR, methylation microarray analysis and qPCR, RNA-seq analysis. Glucose-response genes such as IL10 were subsequently activated. Through IL10 and its receptor IL-10R on tumor cells, macrophages enhanced the in vitro migration of tumor cells in trans-well migration assay verified by shRNA knockdown of IL10 and IL10R respectively. Exogenous infusion of both M1 and M2 macrophages promoted PDA metastasis in orthotopic mouse model after resident macrophage depletion, although pretreating with DNA demethylating agent Decitabine prevented M1, but not M2 macrophages in promoting metastasis, suggesting M1 macrophages were reprogrammed metabolically and functionally by tumor cells to become pro-cancerous. To interrogate the mechanism that regulates the tumor-induced DNA methylation in macrophages, we first examined chromatin remodeling complex Polycomb Repressive Complex 2 (PRC2), with its major component enhancer of zeste homolog 2 (EZH2) previously shown to recruit DNA methyltransferases to specific chromatin regions for epigenetic gene regulation. We found EZH2 protein was most significantly increased in M1 macrophages while Ser21 phosphorylated EZH2 (pSer21-EZH2) remained constant, indicating a decreased percentage of pSer21-EZH2/EZH2 in M1 macrophages co-cultured with tumor cells. Further identification of potential F-box containing ubiquitin ligases that interact with pSer21-EZH2 by Co-immunoprecipitation suggested EZH2 may be stabilized in M1 macrophages by tumor cells through inhibiting the ubiquitin-dependent proteolysis of pSer21-EZH2 in a phosphorylation dependent manner. This study thus far reveals a novel mechanism that reprograms M1 macrophages phenotypically into M2-like macrophages and functionally into pro-cancerous macrophages. Identification of cell surface receptors that mediate the direct tumor-macrophage contact are actively being sought, which will lead to the development of therapeutic blockades to reverse the macrophage reprogramming. Citation Format: Xingyi Pan, Mengwen Zhang, Kenji Fujiwara, Noelle R. Jurcak, Stephen Muth, Lei Zheng. Tumor-associated macrophages are reprogrammed by pancreatic ductal adenocarcinoma cells through tumor-induced DNA methylation on metabolic genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4517.

Macrophage-expressed CD51 promotes cancer stem cell properties via the TGF-β1/smad2/3 axis in pancreatic cancer.

Macrophage-targeted therapy offers new options for intractable pancreatic ductal adenocarcinoma (PDAC), which has a low 5-year survival rate. However, the factors regulating the biological function and phenotype of macrophages in PDAC are incompletely understood. Here, we found that CD51 was positively associated with the poor prognosis of PDAC patients and was highly expressed on macrophages but not on pancreatic cancer cells. Subsequently, we found that CD51 was a marker of macrophages, which promoted the stemness of pancreatic cancer cells. Furthermore, knockdown of CD51 in macrophages drove macrophages toward an M1-like phenotype. Mechanistically, macrophage-expressed CD51 contributed to the acquisition of stemness traits of pancreatic cancer cells by regulating the TGF-β1/smad2/3 pathway. Our data demonstrate the central role played by macrophage-expressed CD51 in the acquisition of stemness traits of pancreatic cancer cells through the paracrine induction of TGF-β1. We first show the connection between the CD51/TGF-β1/smad2/3 axis and PDAC cancer stem cell properties and then indicate that CD51-targeted therapy is a new therapeutic modality for PDAC.

Macrophage CD51 Promotes Cancer Stem Cell Properties via TGFβ1/smad2/3 Axis in Pancreatic Cancer

Background: Targeting macrophage therapy offers new options for intractable pancreatic ductal adenocarcinoma (PDAC) with a low 5-year survival rate. However, the regulators of macrophages in PDAC are not fully understood. Methods: To investigate the relationship between CD51 and PDAC, survival analysis and immunohistochemistry (IHC) were performed. Furthermore, the biomarkers of macrophage polarization were tested to determine the relationship between CD51 and macrophage polarization. Finally, we studied the effects of macrophage CD51 on pancreatic cancer stem cells in vitro and in vivo using functional assays and a xenograft model. Findings: CD51 was positively associated with the poor prognosis of PDAC patients and highly expressed on macrophages, but not on cancer cells. Subsequently, by CD51 knockdown, we found that CD51 was a biomarker of macrophages, which promoted the stemness of pancreatic cancer. Furthermore, knockdown of CD51 in macrophages drove macrophages toward M1-like phenotype. Mechanistically, the CD51 on macrophage contributes to achievement of stemness traits of PDAC by regulating the TGF-β1 expression level. Interpretation: Our data demonstrate the central role that macrophage CD51 plays in the achievement of stemness traits of pancreatic cancer through paracrine induction of TGF-β1. We firstly proved the connection between the CD51/TGF-β1/smad2/3 axis and PDAC cancer stem cell properties and indicated that targeting CD51 therapy represents a new therapeutic modality for PDAC. Funding Statement: This research was supported by grants from the National Natural Science Foundation of China (grant number 81871945 81702951, 81672395, 81672807, 81370059, 81000917, 81702417, and 81402213, www.nsfc.gov.cn), the Guangdong Science and Technology Department (grant number S2012010008934,2014A030313044, 2014A030311047, 2016A030313340, 2016A030313296, and2017A030313880, 2017B030314026 http://pro.gdstc.gov.cn) and the Sun Yat-sen University Clinical Research Foundation of 5010 Project (grant number 2012007). Grant (2013)163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: The authors declare no conflicts of interests. Ethics Approval Statement: The studies using human PDAC tissue samples were approved by the ethics committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University. The protocols for animal studies were approved by the Institutional Animal Care and Use Committee and Institutional Biosafety Committee of Sun Yat-sen University.

Tracking Macrophage Infiltration in a Mouse Model of Pancreatic Cancer with the Positron Emission Tomography Tracer [11C]PBR28

BackgroundThe tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contains abundant immunosuppressive tumor-associated macrophages. High level of infiltration is associated with poor outcome and is thought to represent a major roadblock to lymphocyte-based immunotherapy. Efforts to block macrophage infiltration have been met with some success, but noninvasive means to track tumor-associated macrophagess in PDAC are lacking. Translocator protein (TSPO) is a mitochondrial membrane receptor which is upregulated in activated macrophages. We sought to identify if a radiotracer-labeled cognate ligand could track macrophages in PDAC. Materials and MethodsA murine PDAC cell line was established from a transgenic mouse with pancreas-specific mutations in KRAS and p53. After confirming lack of endogenous TSPO expression, tumors were established in syngeneic mice. A radiolabeled TSPO-specific ligand ([11C] peripheral benzodiazepine receptor [PBR]28) was delivered intravenously, and tumor uptake was assessed by autoradiography, ex vivo, or micro-positron emission tomography imaging. ResultsResected tumors contained abundant macrophages as determined by immunohistochemistry and flow cytometry. Immunoblotting revealed murine macrophages expressed TSPO with increasing concentration on activation and polarization. Autoradiography of resected tumors confirmed [11C]PBR28 uptake, and whole mount sections demonstrated the ability to localize tumors. To confirm the findings were macrophage specific, experiments were repeated in CD11b-deficient mice, and the radiotracer uptake was diminished. Micro-positron emission tomography imaging validated radiotracer uptake and tumor localization in a clinically applicable manner. ConclusionsAs new immunotherapeutics reshape the PDAC microenvironment, tools are needed to better measure and track immune cell subsets. We have demonstrated the potential to measure changes in macrophage infiltration in PDAC using [11C]PBR28.

Abstract 116: IGFBP2 promotes tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most lethal and aggressive malignancy. Tumor-associated macrophages (TAMs) mainly represent the M2-like phenotype, with potent immunosuppressive activity, and play a pro-tumor role in nearly all aspects of PDAC biology. Elevated infiltration of M2 TAMs is associated with poor prognosis in PDAC. Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogene that induces PDAC progression and is involved in the regulation of immune responses. Our recent study demonstrated that IGFBP2 potentiates signal transducer and activator of transcription 3 (STAT3) transactivation activities in glioma. Considering the essential role of IGFBP2 in governing tumor progression and immune evasion of PDAC, we hypothesized that IGFBP2 promotes tumor progression by inducing alternatively activated macrophages in PDAC. In this study, immunohistochemical (IHC) staining for human PDAC clinical samples was used to reveal the relationship among IGFBP2 expression, M2 TAM accumulation and patient survival. The function of IGFBP2 in inducing alternative polarization of macrophages and promoting tumor progression in PDAC through the STAT3 pathway were evaluated using in vitro and in vivo assays. Ingenuity pathway and gene set enrichment analyses were used to identify the correlation of IGFBP2 gene expression with all other genes in the genome and enriched pathways associated with IGFBP2 gene expression. Our results provide evidence that IGFBP2 expression is associated with M2 macrophage accumulation in the tumor microenvironment and with poor prognosis in PDAC. IGFBP2 augments the production and secretion of IL-10 through activation of STAT3 in PDAC cells, thus reprogramming the TAMs polarization toward an M2 phenotype and promoting tumor progression. These findings established IGFBP2 as an immune-suppressor in the PDAC microenvironment and suggested a strategy for targeting IGFBP2 to improve PDAC immunotherapy. Citation Format: Longhao Sun, Liang Liu, Weijun Tian, Zhixiang Zhang, Ya'an Kang, Huamin Wang, Jason B. Fleming, Boris C. Pasche, Wei Zhang. IGFBP2 promotes tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 116.

Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression

Tumor-associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in-depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue-resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas-resident macrophages originated from embryonic development and expanded through insitu proliferation during tumor progression. Whereas monocyte-derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro-fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment.