Pancreatic Ductal Adenocarcinoma Tissues Research Articles

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both invitro and invivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

AHNAK2 Promotes the Progression of Pancreatic Ductal Adenocarcinoma by Maintaining the Stability of c-MET.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and rapidly progresses. The overall response rate of PDAC to current treatment methods is still unsatisfactory. Thus, identifying novel targets and clarifying the underlying mechanisms associated with PDAC progression may potentially offer additional treatment strategies. AHNAK2 is aberrantly expressed in a variety of tumors and exerts pro-tumorigenic effects. However, the biological role of AHNAK2 in PDAC remains poorly understood. The expression of AHNAK2 in PDAC and paired non-tumor tissues was detected by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). Lentivirus knockdown was performed to investigate the impact of AHNAK2 on the biological function of pancreatic cancer cells. The subcutaneous cell-derived xenograft (CDX) model and the KPC spontaneous mouse model with AHNAK2 silencing were used to observe the effects of AHNAK2 on tumor growth and prognosis. The expression of c-MET at protein level in response to HGF treatment was assessed using western blot. Our results demonstrated that AHNAK2 was highly expressed in PDAC clinical samples and associated with poor prognosis. Knockdown of AHNAK2 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells. AHNAK2 knockdown or knockout resulted in tumor growth suppression and prolonged survival in mice with PDAC. In addition, AHNAK2 and c-MET expression levels showed a significant positive correlation at the post-transcriptional level. Mechanistically, AHNAK2 promoted tumor progression by preventing c-MET degradation and persistently activating the HGF/c-MET signaling pathway. Overall, our study revealed that AHNAK2 plays an important role in PDAC progression by modulating the c-MET signaling pathway, and targeting AHNAK2 may be an effective therapeutic strategy for PDAC.

E2F transcription factors promote tumorigenicity in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with limited treatment options, illustrating an urgent need to identify new drugable targets in PDACs. Using the similarities between tumor development and normal embryonic development, which is accompanied by rapid cell expansion, we aimed to identify and characterize embryonic signaling pathways that were reinitiated during tumor formation and expansion. Here, we report that the transcription factors E2F1 and E2F8 are potential key regulators in PDAC. E2F1 and E2F8 RNA expression is mainly localized in proliferating cells in the developing pancreas and in malignant ductal cells in PDAC. Silencing of E2F1 and E2F8 in PANC-1 pancreatic tumor cells inhibited cell proliferation and impaired cell spreading and migration. Moreover, loss of E2F1 also affected cell viability and apoptosis with E2F expression in PDAC tissues correlating with expression of apoptosis and mitosis pathway genes, suggesting that E2F factors promote cell cycle regulation and tumorigenesis in PDAC cells. Our findings illustrate that E2F1 and E2F8 transcription factors are expressed in pancreatic progenitor and PDAC cells, where they contribute to tumor cell expansion by regulation of cell proliferation, viability, and cell migration making these genes attractive therapeutic targets and potential prognostic markers for pancreatic cancer.

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

ObjectiveThe dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of...

Effects of transmembrane serine protease 4 on the survival in patients with pancreatic ductal adenocarcinoma undergoing surgery followed by adjuvant chemotherapy.

The transmembrane serine protease 4 (TMPRSS4) gene is upregulated in various human cancers. However, its biological functions in pancreatic ductal adenocarcinoma remain unclear. We examined the expression of TMPRSS4 in pancreatic ductal adenocarcinoma tissues and its correlation with clinicopathological parameters in patients with pancreatic ductal adenocarcinoma who underwent surgery. The TMPRSS4 expression was immunohistochemically examined in 81 PDAC patients with pancreatic ductal adenocarcinoma. We analyzed the association between the TMPRSS4 expression and clinicopathological factors, the recurrence-free survival (RFS), and the overall survival (OS) and examined the effect of TMPRSS4 expression on cell migration and sensitivity to 5-fluorouracil. The expression rate of TMPRSS4 in the samples was 62.9% (51/81). The TMPRSS4 expression was not correlated with any clinicopathological feature. The five-year overall and recurrence-free survival rates were significantly lower in the TMPRSS4-positive group than in the TMPRSS4-negative group. On a multivariate analysis, TMPRSS4 positivity, poorly differentiated histology, and non-adjuvant chemotherapy predicted a poor OS, while TMPRSS4 positivity and poorly differentiated histology predicted a poor RFS. TMPRSS4-silenced pancreatic ductal adenocarcinoma cells showed higher sensitivity to 5-fluorouracil than did the control siRNA-transfected cells. TMPRSS4 can be considered a prognostic factor and therapeutic target for pancreatic ductal adenocarcinoma.

Integration of single-nucleus and exosome RNA sequencing dissected inter-cellular communication and biomarkers in pancreatic ductal adenocarcinoma

BackgroundMounting evidence underscores the importance of cell communication within the tumor microenvironment, which is pivotal in tumor proliferation, invasion, and metastasis. Exosomes play a crucial role in cell-to-cell communication. Although single-cell RNA sequencing (scRNA-seq) provides insights into individual cell transcriptional characteristics, it falls short of comprehensively capturing exosome-mediated intercellular communication. MethodWe analyzed Pancreatic Ductal Adenocarcinoma (PDAC) tissues, separating supernatant and precipitate for exosome purification and single-cell nucleus suspension. We then constructed Single-nucleus RNA sequencing (snRNA-seq) and small RNA-seq libraries from these components. Our bioinformatic analysis integrated these sequences with ligand-receptor analysis and public miRNA data to map the cell communication network. ResultsWe established intercellular communication networks using bioinformatic analysis to track exosome miRNA effects and ligand-receptor pairs. Significantly, hsa-miR-1293 emerged as a prognostic biomarker for pancreatic cancer, linked to immune evasion, increased myeloid-derived suppressor cells, and poorer prognosis. Targeting this miRNA may enhance anti-tumor immunity and improve outcomes. ConclusionOur study offers a novel approach to constructing intercellular communication networks using snRNA-seq and exosome-small RNA sequencing. By integrating miRNA tracing with ligand-receptor analysis, we illuminate the complex interactions in the pancreatic cancer microenvironment, highlighting the pivotal role of miRNAs and identifying potential biomarkers and therapeutic targets.

Identifying the Spatial Architecture That Restricts the Proximity of CD8+ T Cells to Tumor Cells in Pancreatic Ductal Adenocarcinoma.

The anti-tumor function of CD8+ T cells is dependent on their proximity to tumor cells. Current studies have focused on the infiltration level of CD8+ T cells in the tumor microenvironment, while further spatial information, such as spatial localization and inter-cellular communication, have not been defined. In this study, co-detection by indexing (CODEX) was designed to characterize PDAC tissue regions with seven protein markers in order to identify the spatial architecture that regulates CD8+ T cells in human pancreatic ductal adenocarcinoma (PDAC). The cellular neighborhood algorithm was used to identify a total of six conserved and distinct cellular neighborhoods. Among these, one unique spatial architecture of CD8+ T and CD4+ T cell-enriched neighborhoods enriched the majority of CD8+ T cells, but heralded a poor prognosis. The proximity analysis revealed that the CD8+ T cells in this spatial architecture were significantly closer to themselves and the CD4+ T cells than to the tumor cells. Collectively, we identified a unique spatial architecture that restricted the proximity of CD8+ T cells to tumor cells in the tumor microenvironment, indicating a novel immune evasion mechanism of pancreatic ductal adenocarcinoma in a topologically regulated manner and providing new insights into the biology of PDAC.

Abstract LB316: Endocrine pancreas alteration in pancreatic ductal adenocarcinoma

Abstract Background: The pancreas is central to energy homeostasis, playing essential roles in digestion and regulation of blood glucose. Exocrine pancreas contains large amounts of of hydrolytic enzyme which is responsible for the digestion. Acinar cells, as well as ductal cells, represent the prime components of exocrine pancreas. They are both supposed to be the origin of pancreatic ductal adenocarcinoma(PDAC) and the researches focused on these exocrine cells, as well as other components in pancreatic cancer, are numerous. Islet cells are crucial for glucose homeostasis and their identity are proved to be altered significantly during diabetes by single-cell RNA-sequencing(scRNA-seq). However, the presence or extent of variation of islet cells in PDAC is not established yet. Moreover, due to the low proportion of endocrine cells in pancreas, it is hard to analyze the data of endocrine cells by using published scRNA-seq data of PDAC. As a consequence of this underrepresentation, the function of endocrine cells in PDAC remains unclear. Methods: We used scRNA-seq, multiplex immunohistochemistry (mIHC), high-throughput spatial transcriptomic sequencing (Stereo-seq) to detect the alteration of endocrine pancreas in PDAC of human and mouse model. KPC mice (KrasLSL-G12Dp53LoxPPdx1-CreER) and pancreatic orthotopic transplantation mice were applied to verify the islet cell alteration in multiple stages of PDAC. We also performed islet isolation and purification to enhance the proportion of endocrine cells when preparing single-cell suspension. Cell co-culture experiments were used to determine the interaction between exocrine-endocrine compartments. Results: Through mIHC of islet cells in PDAC tissues and normal pancreas tissues of mice (KPC vs. C57BL/6J) as well as human (PDAC patients vs. healthy donors), we found the decreased proportions of inulin positive (INS+) cells and increased proportions of glucagon positive (GCG+) cells in PDAC tissues. Furthermore, the proportion of pancreatic polypeptide positive (PPY+) cells was significantly elevated in PDAC tissue in both human and murine. Then, we quantified the double-positive cells and found the proportions of INS+GCG+, INS+PPY+ and GCG+PPY+ were markedly increased in PDAC tissue. Additionally, through the experiments in mouse model, we found the islet cells altered gradually within the multiple stage of PDAC(normal-PanIN-PDAC). Finally, it was found that PDAC cells could induce the transdifferentiation of pancreatic α and β cells into GCG+PPY+ and INS+PPY+ double-positive cells, which further promoted PDAC proliferation in a paracrine-dependent manner and formed a reciprocal interaction. Conclusions: Our study systematically maps the alteration of pancreatic endocrine cells in PDAC and elucidates the potential interaction mechanisms between endocrine and exocrine cells during PDAC carcinogenesis. Meanwhile, it is suggested that pancreatic endocrine cells should be considered as an important component of the PDAC microenvironment. Citation Format: Yuan Chen, Xinpeng Yin, Ruiyuan Xu, Rexiati Ruze, Jianlu Song, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Endocrine pancreas alteration in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB316.

Solute carrier family 16 member 1 as a potential prognostic factor for pancreatic ductal adenocarcinoma and its influence on tumor immunity.

Solute carrier family 16 member 1 (SLC16A1) serves as a biomarker in numerous types of cancer. Tumor immune infiltration has drawn increasing attention in cancer progression and treatment. The objective of our study was to explore the association between SLC16A1 and the tumor immune microenvironment in pancreatic ductal adenocarcinoma (PDAC). Data were obtained from The Cancer Genome Atlas. The xCell web tool was used to calculate the proportion of immune cells according to SLC16A1 expression. To further explore the mechanism of SLC16A1, immunity-related genes were screened from differentially expressed genes through weighted gene coexpression network analysis, examined via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and filtrated using univariate Cox regression and least absolute shrinkage and selection operator regression model combined correlation analysis (P<0.05). Next, CIBERSORT was used to analyze the correlation between immune cells and five important genes. SLC16A1 expression and its clinical role in pancreatic cancer was clarified via immunohistochemical staining experiments. Finally, the effects of SLC16A1 on the results of cancer immunity were evaluated by in vitro experiments. SLC16A1 was overexpressed in PDAC tissues and could be an independent prognostic factor. SLC16A1 was significantly negatively correlated with overall survival and suppressed the tumor immunity of PDAC. In clinic, SLC16A1 expression was significantly positively correlated with tumor progression and poor prognosis. We also found that SLC16A1 could suppress the antitumor ability of CD8+ T cells. SLC16A1 is a biomarker for the prognosis of PDAC and can influence the immune environment of PDAC. These findings provide new insights into the treatment of PDAC.

The inverted U-shaped relationship between epinephrine and pancreatic ductal adenocarcinoma patients' survival with compensation of lymphocyte.

The relationship between epinephrine and cancer can be dose-dependent in invivo study. Whether it is the same in human body still needs verification. We used frozen human pancreatic ductal adenocarcinoma (PDAC) tissues to detect epinephrine content and analyzed its relationship with survival using the K-M method and Cox regression. Disturbance of blood cell count and C-reactive protein and identification of related potent intermediary factors were also analyzed. K-M plot and Cox regression all showed the inverted U-shaped relationship between epinephrine and PDAC survival. Lymphocyte adjustment can increase the HRs of epinephrine for PDAC death by >10%. Epinephrine played an anti-tumor or pro-tumor effect depending on the specific concentration. Circulating lymphocyte count was elevated and might acted as a compensation pathway to reduce the pro-tumor effect of epinephrine to PDAC.

Abstract 5587: Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma

Abstract Background and Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with limited treatment options. There is an urgent need for the identification of novel therapeutic targets for PDAC. The transport of molecular cargo between the nucleus and cytoplasm, facilitated by Ran-GTPase, is a critical process that dividing cells rely on to maintain their growth. Elevated nuclear Ran-GTP levels are produced by the activity of the Ran guanine nucleotide exchange factor (GEF) known as Regulator of Chromosome Condensation 1 (RCC1), which is found in the nucleus bound to chromatin. This creates a Ran-GTP gradient across the nuclear membrane, which maintains proper directional transport of proteins and RNA. To sustain proliferative signaling, cancer cells become reliant on high rates of nucleocytoplasmic transport through dysregulation of transport machinery. Methods: We examined the role of RCC1 in the biology of PDAC. The impact of RCC1 modulation on PDAC growth was evaluated using RNA interference and CRISPR-Cas9 in vitro and in vivo using PDAC cell lines and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) tumor-derived cells. The broader impact of RCC1 silencing on PDAC-sustaining signaling was evaluated through RNA-sequencing and proteomics. Results: Transcriptomic sequencing of PDAC tissue (n=5,071) as well as analysis of publicly available data from the TCGA, CPTAC and GTEx revealed that RCC1 expression is higher in PDAC tissues compared to normal pancreas. Moreover, PDAC patients with higher RCC1 expression were more likely to have poorer outcomes. RCC1 silencing by RNAi and CRISPR-Cas9 resulted in reduced proliferation in 2D and 3D cultures and attenuated tumor growth in vivo. RCC1 KD decreased migration and colony formation, enhanced apoptosis, and altered the cell cycle in human and KPC mouse PDAC cells. Subcutaneous RCC1 KO cell line-derived xenografts show arrested growth. Subcellular Ran distribution was disrupted upon RCC1 KO, suggesting that nuclear Ran concentration is important for PDAC proliferation. Nuclear and cytosolic proteomic analysis revealed altered subcellular proteome in RCC1 KD KPC tumor-derived cells. Altered cytoplasmic protein pathways include several metabolic pathways, PI3K-AKT, and Hedgehog signaling. Nuclear enriched pathways include cell cycle, mitosis, metabolic processes, and RNA processing. RNA-seq of RCC1 KO cells showed widespread transcriptional alterations. Upstream of RCC1, c-MYC activates the RCC1-RAN axis, and RCC1 KO cells show differential sensitivity to c-MYC inhibitors. Finally, RCC1 KD resulted in the sensitization of PDAC cells to Gemcitabine. Molecular characterization of conditional Rcc1fl/fl KO transgenic model to study the role of RCC1 in PDAC in vivo is ongoing. Conclusions: Overall, our results show that RCC1 is involved in the regulation of PDAC growth and is a potential target for therapy. Citation Format: Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Md Hafiz Uddin, Rund Nimri, Eliza Beal, Seongho Kim, Mohammed Najeeb Al Hallak, Bassel El-Rayes, Philip A. Philip, Boris Pasche, Ramzi Mohammad, Asfar S. Azmi. Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5587.

ZNF263 cooperates with ZNF31 to promote the drug resistance and EMT of pancreatic cancer through transactivating RNF126.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attribute to the aggressive local invasion, distant metastasis and drug resistance of PDAC patients, which was strongly accelerated by epithelial-mesenchymal transition (EMT). In current study, we systematically investigate the role of ZNF263/RNF126 axis in the initiation of EMT in PDAC in vitro and vivo. ZNF263 is firstly identified as a novel transactivation factor of RNF126. Both ZNF263 and RNF126 were overexpressed in PDAC tissues, which were associated with multiple advanced clinical stages and poor prognosis of PDAC patients. ZNF263 overexpression promoted cell proliferation, drug resistance and EMT in vitro via activating RNF126 following by the upregulation of Cyclin D1, N-cad, and MMP9, and the downregulation of E-cad, p21, and p27. ZNF263 silencing contributed to the opposite phenotype. Mechanistically, ZNF263 transactivated RNF126 via binding to its promoter. Further investigations revealed that ZNF263 interacted with ZNF31 to coregulate the transcription of RNF126, which in turn promoted ubiquitination-mediated degradation of PTEN. The downregulation of PTEN activated AKT/Cyclin D1 and AKT/GSK-3β/β-catenin signaling, thereby promoting the malignant phenotype of PDAC. Finally, the coordination of ZNF263 and RNF126 promotes subcutaneous tumor size and distant liver metastasis in vivo. ZNF263, as an oncogene, promotes proliferation, drug resistance and EMT of PDAC through transactivating RNF126.

Abstract 7291: Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients

Abstract Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP dependent transmembrane efflux pump that confers to drug resistance. Overexpression of MRP1 has been observed in both pancreatic ductal adenocarcinoma (PDAC) and breast cancer tissues. Numerous proteomics studies have revealed the presence of MRP1 in small extracellular vesicles (sEVs) released from cancer cells. Cancer-derived sEVs are known to be released into the circulation, thus becoming attractive candidates for non-invasive biomarker development. However, whether the MRP1 level in plasma sEVs is indicative of human cancer has not been evaluated. This study examined the MRP1 expression in plasma sEVs derived from patients with PDAC, breast and colon cancer, and age- and gender-matched healthy subjects. The patient plasma samples were obtained primarily from the NCI-sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. Healthy plasma samples were provided by the Oklahoma Blood institute. Human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and BxPC-3, and the pancreatic ductal cell line hTERT-HPNE were tested for MRP1 expression prior to examination of plasma samples. Cellular and plasma sEVs were isolated using the double filtration followed by polymer precipitation method. Western blot was performed to assess the expression level of MRP1 and proteomics was applied to confirm the detection of a truncated form of MRP1 in plasma sEVs. MRP1 expression was detected in the sEVs derived from all three pancreatic cancer cell lines but absent in the sEVs from the HPNE line. Overexpression of a truncated MRP1 protein was detected in plasma sEVs derived from patients with early stage (stage I-IIA) and late stage (III-IV) PDAC, compared with that from matched healthy subjects (n=17 for each group). Similar findings were evident in plasma sEVs from patients with breast cancer (n=7), while the results remained inconclusive in plasma sEVs from patients with colon cancer (n=7). Protein structure analysis indicated that the truncated form of MRP1 likely consists of the regions between the QCRL-1 motif and the C-terminus of the protein. Proteomic analysis detected the peptides derived from the C-terminus region, consistent with the structural analysis. Furthermore, the truncated form of MRP1 can only be detected using antibodies against the epitopes in the C-terminus and QCRL-1 region of MRP1 protein. In summary, we have detected a truncated form of MRP1 protein in plasma sEVs derived from cancer patients, and the level of its expression is significantly elevated in patients with PDAC and breast cancer. Citation Format: Kritisha Bhandari, Jeng Shi Kong, Chao Xu, Ajay Jain, Wei-Qun Ding. Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7291.

Abstract 1593: Intercellular network in immune suppressive microenvironment of pancreatic cancer

Abstract Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and in myeloid cell-dominant type, the immune-related pathways were significantly down-regulated. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a therapy in PDAC. Citation Format: Kazunori Aoki, Hironori Fukuda, Yukihiro Mizoguchi, Kosuke Arai. Intercellular network in immune suppressive microenvironment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1593.

Abstract 1604: Cancer-associated fibroblasts modulate T cell killing activity &amp; phenotype in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive malignancy characterised by its abundant extracellular matrix and diverse stromal cell components such as cancer associated fibroblasts (CAFs). CAFs play a pivotal role in shaping the tumour microenvironment and influencing the behaviour of immune cells, particularly T cells, through various mechanisms. The impact of CAFs on T cells is multifaceted as they can exert both pro- and anti-tumoral effects, influencing the delicate balance between immunosurveillance and immune evasion. In this study, we examined the phenotype and function of T cells when co-cultured with CAFs and Adjacent-normal fibroblasts (ANF) isolated from primary PDAC tissue following surgical resection. Methods: Tumour and juxta-tumoral tissue from patients underwent digestion into a single cell suspension. Subsequently, the isolated fibroblasts were cultured and stained with a 22-plex antibody panel designed for flow cytometry. Additional functional assays involved co-culture of CAFs and ANFs with unstimulated or anti-CD3/CD28 stimulated allogeneic T cells. Cell proliferation rates were quantified through the CFSE assay, while the T cells were simultaneously stained to distinguish CD4+ and CD8+ T cells subpopulations. Additionally, the expression of key co-stimulatory molecules: CD69, NKG2D and DNAM-1 and the co-inhibitory molecule: PD-1 were assessed, providing a thorough examination of the immune dynamics within the tumour microenvironment. Results: Flow Cytometry analysis revealed a distinct expression profile in CAFs compared to ANFs, with CAFs expressing higher levels of FAP and CD29. The CFSE assay further revealed that CAFs induced T cell proliferation of resting T cells with no significant increase in stimulated T cells. Notably, this co-culture led to a highly differentiated phenotype in both unstimulated and stimulated CD4+ and CD8+ T cells, characterised by elevated expression of activating markers (CD69 and NKG2D) and the inhibitory molecule PD-1. These findings suggest a dynamic interplay between fibroblasts and T cells in the tumour microenvironment, influencing both the proliferation and phenotypic aspects of T cell behaviour. Citation Format: Fouzia Zayou, Hayden Pearce, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Cancer-associated fibroblasts modulate T cell killing activity &amp; phenotype in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1604.

Abstract 446: Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration

Abstract Purpose: To define the metabolic alterations associated with malignant progression of intraductal papillary mucinous neoplasm (IPMN). Experimental Procedures: Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) was conducted for spatial characterization of lipid profiles on frozen tissue sections from resected human IPMN patients. The specimen set consisted of either low-grade dysplasia (LG) (n=15), high-grade (HG) dysplasia (n=4), or with an associated pancreatic ductal adenocarcinoma (PDAC) (n=4) as well as 15 PDAC tissues. Utilizing serial sections to the same human IPMN cases analyzed with MALDI-MS imaging, we used the Visium Spatial Gene Expression technology platform to characterize the spatiomolecular underpinnings of LG and HG IPMN. H&amp;E images of the same tissue section used for the Visium workflow, were used to annotate the spots in the dataset covering the epithelial lining of the IPMN lesions (“epilesional” areas) and the spots covering the adjacent microenvironment surrounding the lesion (“perilesiopecific” area). Findings were compared with lipidomic analyses of cystic fluid from 89 IPMN patients. The anti-cancer efficacy of UGT8-IN-1, a selective small molecule inhibitor of ceramide galactosyltransferase (CGT, also known as UGT8), was assessed in vitro and in mutant Kras;Gnas allografts. Results: MALDI/MS-based spatial imaging of human resected IPMN tissues and pancreata from a mutant Kras;Gnas mouse model of IPMN revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Visium spatial transcriptomics data confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1, and FA2H, were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid from 89 patients with IPMN identified several sulfatide species to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Inhibition of sulfatide metabolism via UGT8-IN-1 reduced viability of Kras;Gnas murine cell lines in vitro and attenuated tumor growth in an allograft IPMN model. UGT8-IN-1 treatment resulted in reduced levels of several sulfatide species, accompanied by a concomitant increase in a subset of quantified sulfatides precursor ceramides, accumulation of mitochondrial mass and reactive oxygen species and time-dependent increases in autophagy/mitophagy markers p62/SQSTM1 and LC-3B, suggesting impaired mitophagy. Conclusion: Enhanced sulfatide metabolism is an early alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception. Citation Format: Yihui Chen, Marta Sans, Fredrik Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal Rajapakshe, Mark Hurd, Ricardo León-Letelier, Jody Vykoukal, Jennifer Dennison, Kim-Anh Do, Robert Wolff, Paola Guerrero, Michael Kim, Max Schmidt, Anirban Maitra, Samir Hanash, Johannes Fahrmann. Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 446.

Abstract 1576: The heterogeneity between pancreatic ductal adenocarcinoma with &amp; without lymphovascular invasion

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignant solid tumors, with a five-year survival rate less than 10%. Lymphovascular invasion (LVI) is a poor prognostic indicator for PDAC, yet the difference between tumors with or without LVI is unclear. Methods We used scRNA-seq analysis on the PDAC tissue of 24 patients obtained from the National Genomics Data Center (Project number: PRJCA001063) to compare T-cell, macrophage, &amp; cancer-associated fibroblast (CAF) heterogeneity between LVI-positive &amp; LVI-negative PDAC. To validate the scRNA-seq results, complementary techniques, including multiple immunofluorescence assays &amp; flow cytometry, were used. Additionally, we analyzed the characteristics of LVI-positive PDAC tumor cells. Results LVI-positive PDAC thrives in an immunosuppressive microenvironment. We discovered that CD8+ T cells were more deficient in LVI-positive PDAC than in LVI-negative PDAC. Furthermore, the effective &amp; cytotoxic markers (such as GZMB &amp; IFN-γ) of CD8+ T cells also decreased in LVI-positive PDAC. M2 macrophage polarization was found to be higher in LVI-positive PDAC compared to LVI-negative PDAC. In addition, we discovered a more fibrotic TME signature in LVI-positive PDAC. LVI-positive tumor cells, in particular, had a mesenchymal transition phenotype &amp; a hypoxic microenvironment. Conclusions This study adds to our understanding of the intricate heterogeneity seen in LVI-positive PDAC. It sheds light on the distinct characteristics of LVI-positive PDAC &amp; advances our understanding of its underlying biology. Citation Format: Yiping Zou, Yongjie Xie, Song Gao, Tianxing Zhou, Jun Yu, Jihui Hao. The heterogeneity between pancreatic ductal adenocarcinoma with &amp; without lymphovascular invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1576.

Abstract 1602: Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy marked by a highly immunosuppressive tumor microenvironment (TME). Macrophages are known to promote immunosuppression and exhibit a profibrotic transcriptional profile with potential involvement in extracellular matrix (ECM) remodeling. Previously, we identified hyperactivation of cancer cell intrinsic cyclic AMP response element binding protein 1 (CREB) regulates leukemia inhibitory factor (LIF) to mediate macrophage infiltration and polarization within the TME of PDAC. Subsequently, we observed that disrupting LIF-LIF receptor (LIFR) signaling therapeutically not only diminishes the infiltration of tumor-associated macrophages (TAMs) but also results in a decrease in ECM deposition. This study delves into the potential regulatory pathway through which LIF induces remodeling of the ECM within the PDAC TME. Methods: We targeted LIF-induced macrophage-stromal crosstalk using the LIFR antagonist (EC359) in an orthotopically tumor implanted LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice model of PDAC. Sirius red staining in tumor sections was performed to assess ECM (collagen) deposition. Moreover, we performed RNA transcriptomics-based analysis of recombinant (rLIF) induced bone marrow derived macrophages harvested from syngeneic C57BL/6 mice. The Cancer Genome Atlas (TCGA) pancreatic cancer (PAAD) data set along with single-cell RNA sequencing (scRNA seq) were employed to assess the expression of major downstream targets of LIF. To validate the RNA-seq results, we conducted immunohistochemical analysis (IHC) on mouse PDAC tissue sections and quantitative polymerase (qPCR) analysis on RAW 264 macrophage cell line treated with rLIF or KPC CREBWT PDAC tumor cells conditioned media. Results: We observed a drastic decrease in both TAM infiltration and, of note, ECM deposition in orthotopically implanted KPC pancreas tumor mice tissue treated with EC359 as compared to vehicle. Mechanistically, our RNA-seq analysis unveiled a significant upregulation of Factor XIIIA (FXIIIa), a major downstream target of LIF. Notably, TCGA data revealed that FXIIIa mRNA expression is upregulated across PDAC tumors marked by poor survival outcomes. Moreover, scRNA seq and IHC established that TAMs highly express FXIIIa in the PDAC TME. In exploring the effect of LIF-LIFR signaling in the ECM, mouse RAW 264.7 macrophage lines incubated in high LIF conditions confirmed transcriptional upregulation of ECM-associated proteins such as FXIIIa as well as fibronectin. Conclusion: Our investigation provides valuable insights into the possible mechanism by which LIF triggers ECM remodeling, thereby contributing to the desmoplastic stroma. Significantly, our findings propose that LIF-regulated FXIIIa signaling in macrophages serves as a plausible mediator of the LIF-induced ECM remodeling in PDAC. Citation Format: Nishah Jaferi, Siddharth Mehra, Varun Krishnamoorthy, Sudhakar Jinka, Anna Bianchi, Vanessa T. Garrido, Luis A. Nivelo, Ban Yuguang, Nagaraj S. Nagathihalli. Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1602.

Mitochondrial energy metabolism-related gene signature as a prognostic indicator for pancreatic adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a highly malignant gastrointestinal tumor and is associated with an unfavorable prognosis worldwide. Considering the effect of mitochondrial metabolism on the prognosis of pancreatic cancer has rarely been investigated, we aimed to establish prognostic gene markers associated with mitochondrial energy metabolism for the prediction of survival probability in patients with PAAD. Methods: Gene expression data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases, and the mitochondrial energy metabolism-related genes were obtained from the GeneCards database. Based on mitochondrial energy metabolism score (MMs), differentially expressed MMRGs were established for MMs-high and MMs-low groups using ssGSEA. After the univariate Cox and least absolute and selection operator (LASSO) analyses, a prognostic MMRG signature was used in the multivariate Cox proportional regression model. Survival and immune cell infiltration analyses were performed. In addition, a nomogram based on the risk model was used to predict the survival probability of patients with PAAD. Finally, the expression of key genes was verified using quantitative polymerase chain reaction and immunohistochemical staining. Intro cell experiments were performed to evaluated the proliferation and invasion of pancreatic cancer cells. Results: A prognostic signature was constructed consisting of two mitochondrial energy metabolism-related genes (MMP11, COL10A1). Calibration and receiver operating characteristic (ROC) curves verified the good predictability performance of the risk model for the survival rate of patients with PAAD. Finally, immune-related analysis explained the differences in immune status between the two subgroups based on the risk model. The high-risk score group showed higher estimate, immune, and stromal scores, expression of eight checkpoint genes, and infiltration of M0 macrophages, which might indicate a beneficial response to immunotherapy. The qPCR results confirmed high expression of MMP11 in pancreatic cancer cell lines, and IHC also verified high expression of MMP11 in clinical pancreatic ductal adenocarcinoma tissues. In vitro cell experiments also demonstrated the role of MMP11 in cell proliferation and invasion. Conclusion: Our study provides a novel two-prognostic gene signature-based on MMRGs-that accurately predicted the survival of patients with PAAD and could be used for mitochondrial energy metabolism-related therapies in the future.

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4

BackgroundPancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis.MethodsThe expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People’s Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied.ResultsLINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models.ConclusionWe demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.