Pancreatic Ductal Adenocarcinoma Metastasis Research Articles

Abstract A054: PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that regulates a wide variety of cell signaling pathways, including KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demonstrate that the genetic loss of the specific PP2A subunit, B56a, accelerates the formation of KRAS-driven pancreatic lesions in an in vivo mouse model, implicating this subunit as a critical negative regulator of KRAS phenotypes during PDAC progression. Here, we show that acute PP2A-B56a activation prevents the fusion of macropinosomes with lysosomes, robbing PDAC cells of critical metabolic nutrients and driving cell death. Consistent with these findings, PP2A activation also reduces glutamine levels and suppresses oxidative phosphorylation. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, supporting a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M. Pfeffer, Indiraa Doraivel, Brittany L. Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [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 A054.

Abstract A114: Low-dose carbon monoxide halts pancreatic cancer metastasis

Abstract Pancreatic cancer is projected to become the second leading cause of cancer-related death by 2030. Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of pancreatic cancer cases. Over the last 30 years, standard care of chemotherapy only extends the survival of metastatic pancreatic cancer patients for a few months. Despite years of remarkable innovations in cancer treatment, the 5-year survival rate of PDAC has remained dismal. The recalcitrant nature of PDAC to available treatments highlights an urgent need for novel therapeutics in order to substantially increase survival. Carbon monoxide (CO) is produced endogenously in humans and has been established as an important, biologically active signaling molecule. Accumulating evidence suggests the therapeutic applications of safe, low-dose CO for various diseases. We evaluate the therapeutic effect of CO on pancreatic cancer progression. We show that 250 ppm CO inhibits migration of pancreatic cancer. We demonstrate that low-dose CO suppresses metastasis of PDAC in an orthotopic mouse model of liver metastasis and a spontaneous PDAC metastasis mouse model after the removal of primary tumor xenograft tumors. Mechanistically, low-dose CO blocks transcription of heme importers, leading to diminished intracellular heme levels and a heme-regulated enzyme, cytochrome P4501B1 (CYP1B1). Either supplementing heme or overexpressing CYP1B1 reverses the anti-migration effect of low-dose CO. Altogether, low-dose CO therapy inhibits pancreatic cancer cell migration, prevents disseminated cancer cells from expanding into gross metastases, and improves survival in pre-clinical mouse models of metastasis. Citation Format: Tiantian Zhang, George Zhang, Xiang Chen, Zhengming Chen, Adrian Y. Tan, Anthony Lin, Cheryl Zhang, Guoan Zhang, Jenny Xiang, Erika Hissong, Yao-Tseng Chen, Nancy Du. Low-dose carbon monoxide halts pancreatic cancer metastasis [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 A114.

Abstract PR07: Unravelling the epigenetic landscape of pancreatic cancer: The role of cancer-associated fibroblasts

Abstract Pancreatic cancer is set to become the 3rd deadliest cancer by 2025. Pancreatic ductal adenocarcinoma (PDAC) represents the majority of cases and is the most aggressive subtype, with a 5-year survival rate of around 10% and patients often diagnosed at a metastatic stage. Current treatments offer dismal survival benefit for the patients, highlighting the need for innovative therapeutic strategies. PDAC is characterized by genetic and epigenetic modifications and an abundant tumor microenvironment (TME) largely composed of cancer-associated fibroblasts (CAFs). Multiple populations of CAFs have been described and implicated in PDAC progression, however, little is known regarding the molecular crosstalk between them and the cancer cells. This project aims to investigate how the epigenetic landscape of PDAC is affected by recently described CAF populations. We have optimized a co-culture model consisting of cancer cells and genetically-locked inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) to study the link between the TME and the gene regulatory machinery in PDAC through proteomic and genomic approaches. Firstly, a full proteome analysis revealed the two CAF populations drive expression of different subsets of proteins in PDAC cells, including transcription factors. We then interrogated chromatin accessibility via ATAC-seq, which showed that CAF subsets differentially affect chromatin dynamics in epithelial cells. Transcription factor interactomes were investigated using rapid immunoprecipitation of endogenous proteins (RIME), a method developed in our lab that allows the discovery of protein complexes in a robust manner. We leverage this technique to discover druggable transcription factor interactors. RIME data shows that upon co-culture with each CAF population, distinct interactors are recruited towards FOXA1, a pioneer factor that drives a transcriptional program associated with PDAC metastasis to the liver, and towards FOXA2, which has been shown to play distinct roles in different grades of PDAC. We are now assessing the potential of these interactors as drug candidates in each context. Chromatin immunoprecipitation assays have shown that the CAF populations also affect the binding profile of FOXA1 and FOXA2 to the chromatin. We are currently investigating how CAFs impact tumor progression in vivo using an orthotopic implantation model. Preliminary data suggests that CAF subsets affect the metastatic profile in PDAC, as tumors co-injected with iCAFs metastasize to different organs than those co-injected with myCAFs. Ongoing experiments aim to explore the differences between tumors and metastases at the epigenetic level. Subsequent studies will aim to employ distinct approaches that target tumor progression in each context. PDAC is a highly heterogeneous cancer with several layers of complexity; therefore, a more holistic approach is critical to tackle the disease. Understanding tumor-stroma interactions from an epigenetics standpoint may enable identification of druggable molecules much needed for PDAC patients. Citation Format: Catarina Pelicano, Igor Chernukhin, Lisa Young, Naomi Vranas, Yi Cheng, Joshua Kent, Kamal Kishore, Clive D'Santos, Alasdair Russell, Giulia Biffi, Shalivi V. Rao, Jason S. Carroll. Unravelling the epigenetic landscape of pancreatic cancer: The role of cancer-associated fibroblasts [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 PR07.

Identification of Transcriptomic Signatures of Pancreatic Ductal Adenocarcinoma-Derived Exosomes That Promote Macrophage M2 Polarization and Predict Prognosis: S100A9 Reveals Tumor Progression.

Exosomes play a role in intercellular communication and participate in the interaction between pancreatic ductal adenocarcinoma (PDAC) cells and immune cells. Macrophages can receive tumor cell-derived exosomes to polarize into M2-type macrophages, which can enhance the invasion and metastasis of pancreatic cancer, leading to poor prognosis. However, the mechanism by which pancreatic cancer cell-derived exosomes promote M2-type macrophages is still unclear. M2 macrophage-associated exosome-derived key module genes were identified by differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) analysis using exoRbase 2.0, The Cancer Genome Atlas (TCGA), and The International Cancer Genome Consortium (ICGC) databases. Multivariate Cox regression analysis was used to identify key prognostic genes and obtain regression coefficients to establish prognostic signature. Immune infiltration, tumor mutations, and GSEA among different risk groups were compared. exoRbase 2.0, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), HPA, and TISCH2 databases were used to further analyze the expression pattern of S100A9 in pancreatic cancer. In vitro experiments, cell-derived exosome isolation, quantitative polymerase chain reaction (qPCR), western blot, flow cytometry analysis, cell transfection, transwell assay, and CCK-8 assay were applied to investigate the roles of S100A9 in macrophage M2 polarization and tumor progression. The key genes of PDAC-derived exosomes promoting M2-type macrophage polarization were identified, and a risk score model was established. The risk score is related to the expression of common immune checkpoints, immune score, and stromal score, and the tumor mutational burden and biological function of high- and low-risk groups were also different. S100A9 was positively correlated with M2-type macrophage marker. In addition, scRNA-seq data from the TISCH2 database revealed that S100A9 is predominantly expressed in pancreatic cancer cells and mono/macrophage cells, suggesting that S100A9 in pancreatic cancer cells could be received by macrophages, thereby inducing macrophage polarization. In vitro, we used exosomes from BxPC-3 cell lines to coculture macrophages and found that macrophages were mainly polarized toward M2 type, which further promoted the proliferation and metastasis of PDAC. Our study established a reliable risk score model for PDAC-derived exosomes and M2 macrophages, identified the important role of S100A9 in macrophage M2 polarization, which provides a new strategy for the diagnosis and treatment of PDAC, and strengthened the understanding of the mechanism of tumor development and metastasis.

Neural Wiskott-Aldrich syndrome protein (N-WASP) promotes distant metastasis in pancreatic ductal adenocarcinoma via activation of LOXL2.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis has not been established. In this study, we investigated whether Neural Wiskott-Aldrich syndrome protein (N-WASP) plays a role in distant metastasis of PDAC. We found that N-WASP is markedly expressed in clinical patients with PDAC. Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group. N-WASP was noted to be a novel mediator of epithelial-mesenchymal transition (EMT) via gene expression profile studies. Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion, migration, and EMT. We also observed positive association of lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) with the N-WASP-mediated response, wherein EMT and invadopodia function were modulated. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer. These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function, with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.

Vasohibin-2-Targeting Therapies for the Treatment of Pancreatic Ductal Adenocarcinoma.

As pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and refractory, therapeutic options for this cancer are anticipated worldwide. We isolated vasohihibin-2 (VASH2) and observed its overexpression in various types of cancer. We then noticed that upregulated expression of VASH2 in patients with PDAC resulted in a conspicuous reduction in the postoperative survival period and further revealed that the abrogation of Vash2 expression in pancreatic cancer cells inhibited its growth and metastasis and augmented tumor infiltration of CD8+ cells in the mouse model. We developed VASH2-targeting therapies, 2',4'-BNA-based antisense oligonucleotide targeting VASH2 (VASH2-ASO) as a nucleotide-based therapy, and VASH2-peptide vaccine as an antibody-based therapy. We also showed that the VASH2-peptide vaccine inhibited PDAC metastasis in an orthotopic mouse model. Here, we expanded our analysis of the efficacy of VASH2-targeting therapies for PDAC. VASH2-ASO treatment inhibited the growth of primary tumors by reducing tumor angiogenesis, normalizing tumor vessels, preventing ascites accumulation and distant metastasis to the liver and lungs, and augmenting the infiltration of CD8+ cells in metastatic tumors. VASH2-peptide vaccine did not affect the infiltration of CD8+ cells into tumors. The present study revealed that VASH2-targeting therapies are promising options for the treatment of PDAC. VASH2-ASO therapy can be administered at any stage of PDAC. Because of the increase of CD8+ cell infiltration, the combination therapy with immune checkpoint inhibitors may be an attractive option. The VASH2-peptide vaccine therapy may be useful for preventing metastasis and/or recurrence after successful initial treatment.

EGFR-activated myofibroblasts promote metastasis of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Cancer-associated fibroblasts (CAFs) are recognized potential therapeutic targets, but poor understanding of these heterogeneous cell populations has limited the development of effective treatment strategies. We previously identified transforming growth factor beta (TGF-β) as a main driver of myofibroblastic CAFs (myCAFs). Here, we show that epidermal growth factor receptor/Erb-B2 receptor (EGFR/ERBB2) signaling is induced by TGF-β in myCAFs through an autocrine process mediated by amphiregulin. Inhibition of this EGFR/ERBB2-signaling network in PDAC organoid-derived cultures and mouse models differentially impacts distinct CAF subtypes, providing insights into mechanisms underpinning their heterogeneity. Remarkably, EGFR-activated myCAFs promote PDAC metastasis in mice, unmasking functional significance in myCAF heterogeneity. Finally, analyses of other cancer datasets suggest that these processes might operate in other malignancies. These data provide functional relevance to myCAF heterogeneity and identify a candidate target for preventing tumor invasion in PDAC.

Characterization of tumor-associated endothelial cells and the development of a prognostic model in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a complex tumor microenvironment. Angiogenesis is of paramount importance in the proliferation and metastasis of PDAC. However, currently, there are no well-defined biomarkers available to guide the prognosis and treatment of PDAC. In this study, we investigated the interactions between tumor-associated endothelial cells (TAECs) and tumor cells in PDAC, and identified a specific subset of TAECs characterized by high expression of COL4A1. COL4A1+ endothelial cells interact with tumor cells through the COLLAGEN signaling pathway to promote tumor cell proliferation, migration, and invasion. We also observed activation of HOXD9 in COL4A1+ endothelial cells. Based on these findings, we developed a prognostic model called TaEMS, which accurately predicts patient prognosis. TaEMS identified high-risk patients enriched in cell cycle-related pathways and low-risk patients enriched in focal adhesions, smooth muscle regulation, and immune pathways. Moreover, high-risk patients displayed a reduced level of immune cell infiltration, indicating the presence of a “cold tumor” phenotype. Overall, our study uncovered an intricate crosstalk between TAECs and tumor cells in PDAC, emphasizing the role of HOXD9 and highlighting the potential of TaEMS as a prognostic biomarker for precise therapies.

Unraveling dedifferentiation and metastasis traces in pancreatic ductal adenocarcinoma ductal cells: Insights from single-cell RNA sequencing analysis of ITGB4 and C19orf33

Pancreatic Ductal Adenocarcinoma (PDAC) ranks among the most prevalent gastrointestinal malignancies, with risk factors including smoking, alcohol abuse, diabetes mellitus, obesity, age, family history, and genetic predisposition. Extensive research has focused on unraveling biomarkers and molecular intricacies associated with PDAC. Leveraging data from the Gene Expression Omnibus microarray and single-cell RNA sequencing datasets, our study identified ITGB4 and C19orf33 as potentially differentially expressed genes in PDAC samples when contrasted with non-malignant tissues. Notably, these genes exhibited a strong correlative expression pattern, primarily within ductal cells. Gene Expression Profiling Interactive Analysis corroborated our findings, further confirming the correlation between ITGB4 and C19orf33. Additionally, we conducted experiments involving two pivotal PDAC-related cell lines, MIA PaCa-2 and PANC-1, treated with oxaliplatin and 5-Fluorouracil. We also assessed the expression of these candidate genes in PDAC samples in comparison to adjacent normal tissues. Our findings revealed that C19orf33 is upregulated in PDAC samples, and treatment of PDAC cells with chemotherapeutic agents led to a correlated decrease in the expression of both ITGB4 and C19orf33. These co-expressed and correlated genes are implicated in relevant signaling pathways, suggesting shared biological activities that may contribute to the promotion of metastasis within malignant ductal cells. This study identifies ITGB4 and C19orf33 as key genes potentially shedding light on the molecular mechanisms driving tumorigenesis and metastasis in PDAC. These genes hold promise as potential diagnostic and therapeutic targets, offering valuable insights into the management of this challenging disease.

GATA6-AS1 suppresses epithelial–mesenchymal transition of pancreatic cancer under hypoxia through regulating SNAI1 mRNA stability

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a hypoxic microenvironment, a high rate of heterogeneity as well as a high likelihood of recurrence. Mounting evidence has affirmed that long non-coding RNAs (lncRNAs) participate in the carcinogenesis of PDAC cells. In this study, we revealed significantly decreased expression of GATA6-AS1 in PDAC based on the GEO dataset and our cohorts, and showed that low GATA6-AS1 expression was linked to unfavorable clinicopathologic characteristics as well as a poor prognosis. Gain- and loss-of-function studies demonstrated that GATA6-AS1 suppressed the proliferation, invasion, migration, and epithelial–mesenchymal transition (EMT) process of PDAC cells under hypoxia. In vivo data confirm the suppressive roles of GATA6-AS1/SNAI1 in tumor growth and lung metastasis of PDAC. Mechanistically, hypoxia-driven E26 transformation-specific sequence-1 (ETS1), as an upstream modulatory mechanism, was essential for the downregulation of GATA6-AS1 in PDAC cells. GATA6-AS1 inhibited the expression of fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) eraser, and repressed SNAI1 mRNA stability in an m6A-dependent manner. Our data suggested that GATA6-AS1 can inhibit PDAC cell proliferation, invasion, migration, EMT process and metastasis under hypoxia, and disrupting the GATA6-AS1/FTO/SNAI1 axis might be a viable therapeutic approach for refractory hypoxic pancreatic cancers.

Chemokine Receptor 2 Targeted PET/CT Imaging Distant Metastases in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and treatment-refractory malignancies. The lack of an effective screening tool results in the majority of patients being diagnosed at late stages, which underscores the urgent need to develop more sensitive and specific imaging modalities, particularly in detecting occult metastases, to aid clinical decision-making. The tumor microenvironment of PDAC is heavily infiltrated with myeloid-derived suppressor cells (MDSCs) that express C-C chemokine receptor type 2 (CCR2). These CCR2-expressing MDSCs accumulate at a very early stage of metastasis and greatly outnumber PDAC cells, making CCR2 a promising target for detecting early, small metastatic lesions that have scant PDAC cells. Herein, we evaluated a CCR2 targeting PET tracer (68Ga-DOTA-ECL1i) for PET imaging on PDAC metastasis in two mouse models. Positron emission tomography/computed tomography (PET/CT) imaging of 68Ga-DOTA-ECL1i was performed in a hemisplenic injection metastasis model (KI) and a genetically engineered orthotopic PDAC model (KPC), which were compared with 18F-FDG PET concurrently. Autoradiography, hematoxylin and eosin (H&E), and CCR2 immunohistochemical staining were performed to characterize the metastatic lesions. PET/CT images visualized the PDAC metastases in the liver/lung of KI mice and in the liver of KPC mice. Quantitative uptake analysis revealed increased metastasis uptake during disease progression in both models. In comparison, 18F-FDG PET failed to detect any metastases during the time course studies. H&E staining showed metastases in the liver and lung of KI mice, within which immunostaining clearly demonstrated the overexpression of CCR2 as well as CCR2+ cell infiltration into the normal liver. H&E staining, CCR2 staining, and autoradiography also confirmed the expression of CCR2 and the uptake of 68Ga-DOTA-ECL1i in the metastatic foci in KPC mice. Using our novel CCR2 targeted radiotracer 68Ga-DOTA-ECL1i and PET/CT, we demonstrated the sensitive and specific detection of CCR2 in the early PDAC metastases in two mouse models, indicating its potential in future clinical translation.

Elucidating the Role of miRNA-326 Modulating Hedgehog Signaling in Pancreatic Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies worldwide characterized by poor prognosis. MicroRNAs (miRNAs) function as the key regulators in carcinogenesis and may act as noninvasive biomarkers in various malignancies including PDAC. The present study aimed to elucidate the role of miR-326, a known modulator of hedgehog (Hh) pathway in PDAC. miR-326 circulating levels were assessed in 105 PDAC patients, 31 with chronic pancreatitis (CP) and 36 healthy controls by quantitative Polymerase chain reaction. The expression of miR-326 and smoothened (SMO) was checked in surgical PDAC tissue. SMO protein expression was analyzed by immunohistochemistry in different groups. Finally, the role of miR-326 as a modulator of Hh pathway was assessed in vitro. Our results demonstrate that miR-326 is downregulated in both blood and tissue of PDAC patients as compared with controls. In contrast, the target gene/protein expression of SMO is upregulated in PDAC. Moreover, the tumor stromal expression of SMO was found to be clinically associated with lymph-node metastasis and vascular encasement in PDAC. Overexpression of miR-326 in Panc1 cell line was found to induce downregulation of SMO suggesting the tumor suppressor role of miR-326 in PDAC. Taken together, miR-326 acts as a tumor suppressor in PDAC by modulating Hh pathway. It may be a promising target for the development of efficient drug therapies for the treatment of PDAC.

HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective therapies. Here, we demonstrate that the transcription factor, homeobox C6 (HOXC6), is overexpressed in most PDACs, and its inhibition blocks PDAC tumor growth and metastasis. HOXC6 transcriptionally activates tumor-promoting kinase MSK1 and suppresses tumor-inhibitory protein PPP2R2B in PDAC. HOXC6-induced PPP2R2B suppression causes mammalian target of rapamycin (mTOR) pathway activation, which facilitates PDAC growth. Also, MSK1 upregulation by HOXC6 is necessary for PDAC growth because of its ability to suppress apoptosis via its substrate DDX17. Combinatorial pharmacological inhibition of MSK1 and mTOR potently suppressed PDAC tumor growth and metastasis in PDAC mouse models. PDAC cells with acquired resistance to MSK1/mTOR-inhibitors displayed activated insulin-like growth factor 1 receptor (IGF1R) signaling and were successfully eradicated by IGF1R inhibitor. Furthermore, MEK inhibitor trametinib enhanced the efficacy of dual MSK1 and mTOR inhibition. Collectively, these results identify therapeutic vulnerabilities of PDAC and an approach to overcome acquired drug resistance to prolong therapeutic benefit.

Prediction of postoperative liver metastasis in pancreatic ductal adenocarcinoma based on multiparametric magnetic resonance radiomics combined with serological markers: a cohort study of machine learning.

To construct and validate a multi-dimensional model based on multiple machine leaning algorithms to predict PCLM using multi-parameter magnetic resonance (MRI) sequences with clinical and imaging parameters. A total of 148 PDAC retrospectively examined patients were classified as metastatic or non-metastatic based on results at 3 months after surgery. The radiomics features of the primary tumor were extracted from T2WI images, followed by dimension reduction. Then, multiple machine learning methods were used to construct models. Independent predictors were also screened using multifactor logistic regression and a nomogram was constructed in combination with the radiomics model. Area under the receiver operating characteristic curve (AUC) and decision curve analysis (DCA) were used to assess the accuracy and reliability of the nomogram. The diagnostic efficacy of the radiomics model in the training and test set was 0.822 and 0.803, sensitivity was 0.742 and 0.692, and specificity was 0.792 and 0.875, respectively. The diagnostic efficacy of the nomogram in the training and test set was 0.866 and 0.832. A radiomics nomogram based on machine learning improved the accuracy of predicting PCLM and may be useful for early preoperative diagnosis.

Kupffer cells prevent pancreatic ductal adenocarcinoma metastasis to the liver in mice

Although macrophages contribute to cancer cell dissemination, immune evasion, and metastatic outgrowth, they have also been reported to coordinate tumor-specific immune responses. We therefore hypothesized that macrophage polarization could be modulated therapeutically to prevent metastasis. Here, we show that macrophages respond to β-glucan (odetiglucan) treatment by inhibiting liver metastasis. β-glucan activated liver-resident macrophages (Kupffer cells), suppressed cancer cell proliferation, and invoked productive T cell-mediated responses against liver metastasis in pancreatic cancer mouse models. Although excluded from metastatic lesions, Kupffer cells were critical for the anti-metastatic activity of β-glucan, which also required T cells. Furthermore, β-glucan drove T cell activation and macrophage re-polarization in liver metastases in mice and humans and sensitized metastatic lesions to anti-PD1 therapy. These findings demonstrate the significance of macrophage function in metastasis and identify Kupffer cells as a potential therapeutic target against pancreatic cancer metastasis to the liver.

Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma.

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.

Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis.

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

CircEIF3I facilitates the recruitment of SMAD3 to early endosomes to promote TGF-β signalling pathway-mediated activation of MMPs in pancreatic cancer

BackgroundAmong digestive tract tumours, pancreatic ductal adenocarcinoma (PDAC) shows the highest mortality trend. Moreover, although PDAC metastasis remains a leading cause of cancer-related deaths, the biological mechanism is poorly understood. Recent evidence demonstrates that circular RNAs (circRNAs) play important roles in PDAC progression.MethodsDifferentially expressed circRNAs in normal and PDAC tissues were screened via bioinformatics analysis. Sanger sequencing, RNase R and actinomycin D assays were performed to confirm the loop structure of circEIF3I. In vitro and in vivo functional experiments were conducted to assess the role of circEIF3I in PDAC. MS2-tagged RNA affinity purification, mass spectrometry, RNA immunoprecipitation, RNA pull-down assay, fluorescence in situ hybridization, immunofluorescence and RNA–protein interaction simulation and analysis were performed to identify circEIF3I-interacting proteins. The effects of circEIF3I on the interactions of SMAD3 with TGFβRI or AP2A1 were measured through co-immunoprecipitation and western blotting.ResultsA microarray data analysis showed that circEIF3I was highly expressed in PDAC cells and correlated with TNM stage and poor prognosis. Functional experiments in vitro and in vivo revealed that circEIF3I accelerated PDAC cells migration, invasion and metastasis by increasing MMPs expression and activity. Mechanistic research indicated that circEIF3I binds to the MH2 domain of SMAD3 and increases SMAD3 phosphorylation by strengthening the interactions between SMAD3 and TGFβRI on early endosomes. Moreover, AP2A1 binds with circEIF3I directly and promotes circEIF3I-bound SMAD3 recruitment to TGFβRI on early endosomes. Finally, we found that circEif3i exerts biological functions in mice similar to those of circEIF3I in humans PDAC.ConclusionsOur study reveals that circEIF3I promotes pancreatic cancer progression. circEIF3I is a molecular scaffold that interacts with SMAD3 and AP2A1 to form a ternary complex, that facilitates the recruitment of SMAD3 to early endosomes and then activates the TGF-β signalling pathway. Hence, circEIF3I is a potential prognostic biomarker and therapeutic target in PDAC.

Single-cell mapping reveals several immune subsets associated with liver metastasis of pancreatic ductal adenocarcinoma

Identifying a metastasis-correlated immune cell composition within the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) will help to develop promising and innovative therapeutic strategies. However, the dynamics of immune cell lineages in the TME of advanced PDAC remains elusive. Twenty-six samples from 11 patients (including 11 primary tumor tissues, 10 blood, and 5 lymph nodes) with different stages were used to develop a multiscale immune profile. High-dimensional single-cell analysis with mass cytometry was performed to search for metastasis-correlated immune changes in the microenvironment. The findings were further validated by published single-cell RNA sequencing (scRNA-seq) data and multiplex fluorescent immunohistochemistry. High-dimensional single-cell profiling revealed that the three immune-relevant sites formed a distinct immune atlas. Interestingly, thePDAC microenvironment with the potential for metastatic spread to the liver was characterized by a decreased proportion of CD103+PD-1+CD39+ Tcells with cytotoxic and exhausted functional status and an increased proportion of CD73+ macrophages. Analysis of scRNA-seq data of PDAC further confirmed the identified subsets and revealed strong potential interactions via various ligand-receptor pairs between the identified T subsets and the macrophages. Moreover, stratified patients with different immune compositions correlated with clinical outcomes of PDAC. Our study uncovered metastasis-correlated immune changes, suggesting that ecosystem-based patient classification in PDAC will facilitate the identification of candidates likely to benefit from immunotherapy. This work was supported by the National Key Research and Development Program of China, the Shanghai International Science and Technology Collaboration Program, the Shanghai Sailing Program, and the Key Laboratory of diagnosis and treatment of severe hepato-pancreatic diseases of Zhejiang Province.

Increased circulating Th17 cell populations in patients with pancreatic ductal adenocarcinoma.

T-helper 17 (Th17) cells are a subset of CD4+ helper T cells that produce interleukin 17 (IL-17) and play a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Few studies have been conducted to determine the role of Th17 cells in the tumorigenesis and development of pancreatic ductal adenocarcinoma (PDAC); however, its role is still unclear. In this study, the percentage of circulating Th17 cells and serum levels of IL-17A and IL-23 were analyzed using flow cytometry and ELISA, respectively, in 40 PDAC patients, 30 chronic pancreatitis (CP) patients and 30 healthy controls (HC). In addition, the mRNA expression levels of IL-17A, STAT3 and RORγt in tissue samples were quantified by qRT-PCR. The results showed that the percentage of circulating Th17 cells and the concentrations of serum IL-17A and IL-23 were significantly increased in PDAC patients as compared to CP and HC (P < 0.001). In addition, the higher level of IL-17A was significantly correlated with the poor overall survival of the PDAC patients. Furthermore, the frequencies of Th17 cells and IL-17A were significantly higher in stage III+IV PDAC patients versus stage I+II. A significant increase in IL-17A, STAT3 and RORγT mRNA was observed in patients with PDAC. Taken together, these findings suggest that the increased circulating Th17 cells and serum IL-17A may be involved in the development and metastasis of PDAC, and thus represent potential targets for the treatment of PDAC.