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The carbohydrate antigen 19-9 (CA19-9) is commonly used as a representative biomarker for pancreatic cancer (PC); however, it lacks sensitivity and specificity for early-stage PC diagnosis. Furthermore, some patients with PC are negative for CA19-9 (<37 U/mL), which introduces additional limitations to their accurate diagnosis and treatment. Hence, improved methods to accurately detect PC stages in CA19-9-negative patients are warranted. In this study, tumor-proximal liquid biopsy and inertial microfluidics were coupled to enable high-throughput enrichment of portal venous circulating tumor cells (CTCs) and support the effective diagnosis of patients with early-stage PC. The proposed inertial microfluidic system was shown to provide size-based enrichment of CTCs using inertial focusing and Dean flow effects in slanted spiral channels. Notably, portal venous blood samples were found to have twice the yield of CTCs (21.4 cells per 5 mL) compared with peripheral blood (10.9 CTCs per 5 mL). A combination of peripheral and portal CTC data along with CA19-9 results showed to greatly improve the average accuracy of CA19-9-negative PC patients from 47.1% with regular CA19-9 tests up to 87.1%. Hence, portal venous CTC-based microfluidic biopsy can be used with high sensitivity and specificity for the diagnosis of early-stage PC, particularly in CA19-9-negative patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pancreatic cancer (PC) is a highly malignant tumour of the digestive system with poor therapeutic response and low survival rates. Immunotherapy has rapidly developed in recent years and has achieved significant outcomes in numerous malignant neoplasms. However, responses to immunotherapy in PC are rare, and the immunosuppressive and desmoplastic tumour microenvironment (TME) significantly hinders their efficacy in PC. Tumour-associated neutrophils (TANs) play a crucial role in the PC microenvironment and exert a profound influence on PC immunotherapy by establishing a robust stromal shelter and restraining immune cells to assist PC cells in immune escape, which may subvert the current status of PC immunotherapy. The present review aims to offer a comprehensive summary of the latest progress in understanding the involvement of TANs in PC desmoplastic and immunosuppressive functions and to emphasise the potential therapeutic implications of focusing on TANs in the immunotherapy of this deleterious disease. Finally, we provide an outlook for the future use of TANs in PC immunotherapy., (© 2024 John Wiley & Sons Ltd.)

Pancreatic cancer is the most fatal malignancy worldwide. Once diagnosed, most patients are already at an advanced stage because of their highly heterogeneous, drug-resistant, and metastatic nature and the lack of effective diagnostic markers. Recently, the study of proliferation, metastasis, and drug resistance mechanisms in pancreatic cancer and the search for useful diagnostic markers have posed significant challenges to the scientific community. Exosomes carry various biomolecules (DNA, non-coding RNAs (ncRNAs), proteins, and lipids) that mediate communication between tumors and other cells. ncRNAs can be transported through exosomes to numerous relevant receptor cells and regulate local epithelial-mesenchymal transition (EMT) in tumor tissue, proliferation, drug resistance, and the establishment of pre-metastatic ecological niches in distant organs. In summary, exosomal ncRNAs promote tumor cell proliferation, invasion, and metastasis through multiple EMT, immunosuppression, angiogenesis, and extracellular matrix remodeling pathways. Moreover, we discuss the significant therapeutic significance of exosomal ncRNAs as PC biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lipid peroxidation-dependent ferroptosis has become an emerging strategy for tumor therapy. However, current strategies not only selectively induce ferroptosis in malignant cells but also trigger ferroptosis in immune cells simultaneously, which can compromise anti-tumor immunity. Here, we used In-Cell Western assays combined with an unbiased drug screening to identify the compound N6F11 as a ferroptosis inducer that triggered the degradation of glutathione peroxidase 4 (GPX4), a key ferroptosis repressor, specifically in cancer cells. N6F11 did not cause the degradation of GPX4 in immune cells, including dendritic, T, natural killer, and neutrophil cells. Mechanistically, N6F11 bound to the RING domain of E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) in cancer cells to trigger TRIM25-mediated K48-linked ubiquitination of GPX4, resulting in its proteasomal degradation. Functionally, N6F11 treatment caused ferroptotic cancer cell death that initiated HMGB1-dependent antitumor immunity mediated by CD8 + T cells. N6F11 also sensitized immune checkpoint blockade that targeted CD274/PD-L1 in advanced cancer models, including genetically engineered mouse models of pancreatic cancer driven by KRAS and TP53 mutations. These findings may establish a safe and efficient strategy to boost ferroptosis-driven antitumor immunity.

Ferroptosis is a form of regulated cell death characterized by oxidative injury-induced lipid peroxidation. However, the detailed protein post-translational modification regulatory mechanism of ferroptosis remains largely unknown. Here, we report that E1A binding protein P300 (EP300) acetyltransferase promotes ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells via the acetylation of heat shock protein family A (Hsp70) member 5 (HSPA5), also known as GRP78 or BIP) on the site of K353. Acetylated HSPA5 loses its ability to inhibit lipid peroxidation and subsequent ferroptotic cell death. Genetic or pharmacological inhibition of EP300-mediated HSPA5 acetylation on K353 increases PDAC cell resistance to ferroptosis. Moreover, histone deacetylase 6 (HDAC6) limits HSPA5 acetylation and subsequent ferroptosis. Collectively, these findings not only identify regulatory pathways for HSPA5 acetylation during ferroptosis, but also highlight promising strategies to increase ferroptosis sensitivity in PDAC cells., (© 2023. Springer Nature Limited.)

Aims: Ferroptosis, a type of oxidative cell death driven by unlimited lipid peroxidation, is emerging as a target for cancer therapy. Although mitochondrial dysfunction may lead to ferroptosis, the underlying molecular mechanisms and metabolic pathways for ferroptosis are incompletely understood. Here, we identify solute carrier family 25 member 22 (SLC25A22), a mitochondrial glutamate transporter, as a driver of ferroptosis resistance in pancreatic ductal adenocarcinoma (PDAC) cells. Results: The downregulation of SLC25A22 expression was associated with increased sensitivity to ferroptosis, but not to apoptosis. Mechanistically, on the one hand, SLC25A22-dependent NAPDH synthesis blocks ferroptotic cell death in PDAC cells through mediating the production of glutathione (GSH), the most important hydrophilic antioxidant. On the other hand, SLC25A22 promotes the expression of stearoyl-CoA desaturase in PDAC cells in an AMP-activated protein kinase-dependent manner, resulting in the production of antiferroptotic monounsaturated fatty acids (MUFAs). The animal study further confirms that SLC25A22 inhibits ferroptosis-mediated tumor suppression. Innovation: SLC25A22 is a novel metabolic repressor of ferroptosis by producing GSH and MUFAs. Conclusion: These findings establish a previously unrecognized metabolic defense pathway to limit ferroptotic cell death in vitro and in vivo . Antioxid. Redox Signal. 39, 166-185.

Pancreatic cancer is an increasingly growing source of cancer-related deaths and is often diagnosed at advanced stages. Its treatment is difficult because of the poor results of conventional treatments, such as surgery, chemotherapy, and radiotherapy. Microbiota and their products can regulate the microenvironment of pancreatic tumors, the biological behavior of pancreatic cancer cells, and the functionality of the immune system. Promising results have been achieved in treating pancreatic cancer by regulating microbiota. However, intratumoral microbiota is still in its infancy as a new field of discovery for pancreatic cancer. This review summarizes the mechanisms by which intratumoral microbiota causes pancreatic cancer tumorigenesis, progression, and metastasis and demonstrates their significant potential in diagnosing and treating pancreatic cancer. Additionally, we present an outlook on the future of intratumoral microbiota in treating pancreatic cancer., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Alkaliptosis, a type of regulated cell death driven by intracellular alkalization, was first described in pancreatic ductal adenocarcinoma (PDAC) cells after treatment with the opioid analgesic drug JTC801. Here, we used mass-spectrometry-based drug target identification, cellular thermal shift assay, and point mutation technologies to reveal ATP6V0D1 as a direct JTC801 target that drives alkaliptosis in human PDAC cells. Functionally, the protein stability of ATP6V0D1, when mediated by JTC801, increases the interaction between ATP6V0D1 and STAT3, resulting in increased expression and activity of STAT3 for sustaining lysosome homeostasis. Consequently, the pharmacological or genetic inhibition of STAT3 restores the sensitivity of ATP6V0D1-deficient cells to alkaliptosis in vitro or in suitable mouse models. Clinically, a high expression of ATP6V0D1 correlates with prolonged survival of patients with PDAC. Together, these results illustrate a link between ATP6V0D1 and PDAC and advance our understanding of alkaliptosis in targeted therapy., Competing Interests: Declaration of interests The authors declare no conflicts of interest or financial interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alkaliptosis is a recently discovered type of pH-dependent cell death used for tumor therapy. However, its underlying molecular mechanisms and regulatory networks are largely unknown. Here, we report that the acetate-activating enzyme acetyl-CoA short-chain synthase family member 2 (ACSS2) is a positive regulator of alkaliptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Using qPCR and western blot analysis, we found that the mRNA and protein expression of ACSS2 was upregulated in human PDAC cell lines (PANC1 and MiaPaCa2) in response to the classic alkaliptosis activator JTC801. Consequently, the knockdown of ACSS2 by shRNAs inhibited JTC801-induced cell death in PDAC cells, and was accompanied by an increase in cell clone formation and a decrease in intracellular pH. Mechanically, ACSS2-mediated acetyl-coenzyme A production and subsequent histone acetylation contributed to NF-κB-dependent CA9 downregulation, and this effect was enhanced by the histone deacetylase inhibitor trichostatin A. These findings may provide new insights for understanding the metabolic basis of alkaliptosis and establish a potential strategy for PDAC treatment., (© 2023. The Author(s).)

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC., Methods: Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for in vitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth in vivo. Mouse PDAC cells derived from the Kras LSL-G12D/+ ;Tp53 L/+ ;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis in vivo., Results: High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration in vitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin β4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin β4 protein, which may have accounted for the suppression of metastasis., Conclusions: Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Three-dimensional (3D) organoid culture is a laboratory technique used to grow and study miniature organs that mimic the structure and function of real organs in the human body. Organoids are created from stem cells or tissue samples and are grown in a 3D matrix that allows them to self-organize into a complex, three-dimensional structure. Organoids are valuable tools for studying human biology and disease, including cancer. Pancreatic ductal adenocarcinoma (PDAC) still has the worst survival rate of common malignancies, despite recent advances in cancer treatment. Preclinical studies have shown that impaired cell death pathways, including apoptosis, necroptosis, ferroptosis, pyroptosis, and alkaliptosis, promote PDAC development. Organoid models are now widely used in the study of pancreatic cancer biology, including cell death machinery. This chapter provides step-by-step protocols for generating human or mice PDAC organoids in a 3D Matrigel system., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immunotherapy has shown promising efficacy in the treatment of a wide range of cancers; however, it has had little effect on pancreatic cancer. Cancer-associated fibroblasts (CAFs), the predominant mesenchymal cells present in the pancreatic cancer microenvironment, are powerful supporters of the malignant progression of pancreatic cancer. CAFs can modify the microenvironment, establish a refuge to aid cancer cells in immune escape by secreting large amounts of extracellular matrix, and produce soluble cytokines and exosomal vesicles. Hence, CAFs are important contributors to the failure of immunotherapy. Current in-depth studies of CAFs have shown that CAFs are a heterogeneous population of mesenchymal cells; therefore, the functional complexity of their populations needs in-depth explorations in future studies. This review summarizes how heterogeneous CAFs help cancer cells achieve immune escape and suggests potential directions for using CAFs as targets to address immune escape., (© 2022 John Wiley & Sons Ltd.)

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal gastrointestinal cancer, and its poor prognosis is highly associated with the lack of an efficient early detection technology. Here, we report that RGD-NGR heterodimer labeled with PET isotope could be applied in PDAC early detection., Procedures: The RGD-NGR tracer was first compared with its corresponding monomeric counterparts via PET imaging studies using mice bearing a subcutaneous BxPC3 tumor. Subsequently, the RGD-NGR tracer was evaluated in autochthonous mouse models with spontaneously developed late stage PanIN lesions (KC ER mice) or PDAC (KPC mice) via both PET imaging studies and ex vivo biodistribution studies. Furthermore, a comparison between 2-deoxy-2[ 18 F]fluoro-D-glucose ([ 18 F]F-FDG) and the RGD-NGR tracer was conducted via PET imaging of the same KCH mouse bearing spontaneously developed PDAC. H&E staining was performed to confirm the malignant pancreatic tissue in the KCH mouse. Immunofluorescence staining was performed to confirm the expression of integrin α V β 3 and CD13., Results: The RGD-NGR tracer exhibited improved in vivo performance as compared with its corresponding monomeric counterparts on the subcutaneous BxPC3 tumor mouse model. Subsequent evaluation in autochthonous mouse models demonstrated its capability to detect both pre-malignant and malignant pancreases. Further comparison with [ 18 F]F-FDG revealed the superiority of the proposed heterodimer in imaging spontaneously developed PDAC. H&E staining confirmed the malignant pancreatic tissue in the KCH mouse, while the expression of both integrin α V β 3 and CD13 receptors was demonstrated with immunofluorescence staining., Conclusion: The proposed RGD-NGR heterodimer possesses the potential to be applied in the PDAC early detection for high-risk populations., (© 2022. World Molecular Imaging Society.)

Background: Endoscopic ultrasound (EUS) is widely used as a cost-effective method for detecting pancreatic neuroendocrine tumors (PNTs), but its diagnostic value is variable among published studies. This meta-analysis aimed to determine the diagnostic value of EUS for PNTs., Methods: Three electronic databases, including PubMed, Embase, and the Cochrane Library, were searched for studies published up to July 2018. The summary sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (ROC) curve were calculated to evaluate the diagnostic value of EUS using the random-effects model., Results: Thirteen studies involving 609 patients were included in this meta-analysis. The summary sensitivity and specificity of EUS for detecting PNTs were 0.86 and 0.89, respectively. The PLR and NLR of EUS were 7.81 and 0.15, respectively. The DOR of EUS for diagnosing PNTs was 24.20. The area under the ROC was 0.90. Finally, the subgroup analyses indicated that publication year and percentage of males could introduce potential biases for the DOR of EUS., Conclusions: This meta-analysis suggests that EUS has a relatively high diagnostic value for diagnosing PNTs., (Copyright © 2022 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Background: We aimed to develop a deep learning-based segmentation system for rapid on-site cytopathology evaluation (ROSE) to improve the diagnostic efficiency of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) biopsy., Methods: A retrospective, multicenter, diagnostic study was conducted using 5345 cytopathological slide images from 194 patients who underwent EUS-FNA. These patients were from Nanjing Drum Tower Hospital (109 patients), Wuxi People's Hospital (30 patients), Wuxi Second People's Hospital (25 patients), and The Second Affiliated Hospital of Soochow University (30 patients). A deep convolutional neural network (DCNN) system was developed to segment cell clusters and identify cancer cell clusters with cytopathological slide images. Internal testing, external testing, subgroup analysis, and human-machine competition were used to evaluate the performance of the system., Findings: The DCNN system segmented stained cells from the background in cytopathological slides with an F1-score of 0·929 and 0·899-0·938 in internal and external testing, respectively. For cancer identification, the DCNN system identified images containing cancer clusters with AUCs of 0·958 and 0·948-0·976 in internal and external testing, respectively. The generalizable and robust performance of the DCNN system was validated in sensitivity analysis (AUC > 0·900) and was superior to that of trained endoscopists and comparable to cytopathologists on our testing datasets., Interpretation: The DCNN system is feasible and robust for identifying sample adequacy and pancreatic cancer cell clusters. Prospective studies are warranted to evaluate the clinical significance of the system., Funding: Jiangsu Natural Science Foundation; Nanjing Medical Science and Technology Development Funding; National Natural Science Foundation of China., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Microbes and their metabolites are found in all body organs; their interaction with body organs can influence inflammation, immunity, and cancer development. Pancreatic cancer development is closely related to intestinal, intrapancreatic, and oral flora. The microbiota plays a regulatory role in pancreatic cancer's malignant progression and treatment resistance. Thus, the study of microbiota-host interactions has emerged as a new hot topic in pancreatic cancer treatment, with microbiota control demonstrating significant clinical potential. This review summarizes recent advances in the clinical diagnosis and treatment of pancreatic cancer, emphasizing the enormous potential for operating microbiota in pancreatic cancer treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pancreatic cancer is one of the most common malignancies. Unfortunately, the lack of effective methods of treatment and diagnosis has led to poor prognosis coupled with a very high mortality rate. So far, the pathogenesis and progression mechanisms of pancreatic cancer have been poorly characterized. Exosomes are small vesicles secreted by most cells, contain lipids, proteins, and nucleic acids, and are involved in diverse functions such as intercellular communications, biological processes, and cell signaling. In pancreatic cancer, exosomes are enriched with multiple signaling molecules that mediate intercellular communication with control of immune suppression, mutual promotion between pancreas stellate cells and pancreatic cancer cells, and reprogramming of normal cells. In addition, exosomes can regulate the pancreatic cancer microenvironment and promote the growth and survival of pancreatic cancer. Exosomes can also build pre-metastatic micro-ecological niches and facilitate the targeting of pancreatic cancer. The ability of exosomes to load cargo and target allows them to be of great clinical value as a biomarker mediator for targeted drugs in pancreatic cancer. Video Abstract., (© 2022. The Author(s).)

Pancreatic cancer is a highly malignant tumor and, is extremely difficult to diagnose and treat. Metastasis is one of the critical steps in the development of cancer and uses cell to cell communication to mediate changes in the microenvironment. Small extracellular vesicles (sEVs)-carry proteins, nucleic acids and other bioactive substances, and are important medium for communication between cells. There are two primary steps in sVEs-mediated metastasis: communication between pancreatic cancer cells and their surrounding microenvironment; and the communication between primary tumor cells and distant organ cells in distant organs that promotes angiogenesis, reshaping extracellular matrix, forming immunosuppressive environment and other ways to form appropriate pre-metastasis niche. Here, we explore the mechanism of localization and metastasis of pancreatic cancer and use sEVs as early biomarkers for the detection and treatment of pancreatic cancer. Video Abstract., (© 2021. The Author(s).)

Calcium based biomaterials were widely used for drug delivery application due to their biodegradability, biocompatibility, and high drug loading capacity. Herein, amino-capped polyamidoamine (PAMAM) dendrimer was applied as a macromolecular template to form amino-modified calcium phosphate hollow sphere (CaPO-NH 2 ). After loading with 5-fluorouracil (5Fu), this system performed synergistic cancer chemotherapy. In this study, the 5Fu/CaPO-NH 2 particles could be efficiently uptaken by cancer cells, and then decompose into Ca 2+ and release 5Fu drug in the cytoplasm; therefore calcium overload and reactive oxygen species (ROS) accumulation were found in PSN1 cells that could induce cell membrane damage and elicit cell apoptosis through a series of biochemical reactions including endoplasmic reticulum stress, lipid peroxidation and mitochondrial apoptosis. In the PSN1 pancreatic cancer xenograft model, the 5Fu/CaPO-NH 2 system performed high tumor inhibition via chemotherapy and calcium overload induced apoptosis. Comparingly, the normal cells and organs were insensitive to this synergistic therapy, which indicated the well biocompatibility of delivery system. Thus, this study provided a promising CaPO-NH 2 drug delivery platform for enhanced 5Fu chemotherapeutic effect., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Our study seeks to obtain data to assess the impact of circPUM1 on pancreatic cancer (PC) and its mechanism., Methods: The expression of circPUM1 and miR-200c-3p in PC and normal tissues and PC cell lines was collected and detected, and subsequently dual-luciferase assay-based verification of the binding site of the two was carried out. After interfering with circPUM1 expression in MIAPaCa-2 and PANC-1 cells, cell proliferation, viability, apoptosis rate, invasion ability, glucose consumption, and lactate production were measured by MTT, colony formation, flow cytometry, Transwell assays, and glucose and lactate assay kits. Additionally, western blot was utilized for assessing PI3K/AKT signaling pathway-related proteins. From the results, highly expressed circPUM1 and miR-200c-3p in PC tissues and cells were proved., Results: Down-regulation of circPUM1 expression significantly inhibited cell proliferation, cell viability, invasion and glycolysis, while increasing the apoptosis rate. Down-regulated circPUM1 led to the inhibition of the PI3K/AKT signaling pathway activity in PC cells; while up-regulated circPUM1 increased its activity. Further experiments revealed that down-regulation of miR-200c-3p expression reversed the inhibitory effect of lowly expressed circPUM1 on PC cells., Conclusion: In summary, circPUM1 activates PI3K/AKT signaling pathway by sponging miR-200c-3p and promotes PC progression., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Pancreatic cancer is a devastating gastrointestinal cancer characterized by late diagnosis, limited treatment success and dismal prognosis. Exocrine tumours account for 95% of pancreatic cancers and the most common pathological type is pancreatic ductal adenocarcinoma (PDAC). The occurrence and progression of PDAC involve multiple factors, including internal genetic alterations and external inflammatory stimuli. The biology and therapeutic response of PDAC are further shaped by various forms of regulated cell death, such as apoptosis, necroptosis, ferroptosis, pyroptosis and alkaliptosis. Cell death induced by local or systemic treatments suppresses tumour proliferation, invasion and metastasis. However, unrestricted cell death or tissue damage might result in an inflammation-related immunosuppressive microenvironment, which is conducive to tumour progression or recurrence. The precise extent to which cell death affects PDAC is not yet well described. A growing body of preclinical and clinical studies document significant correlations between mutations (for example, in KRAS and TP53), stress responses (such as hypoxia and autophagy), metabolic reprogramming and chemotherapeutic responses. Here, we describe the molecular machinery of cell death, discuss the complexity and multifaceted nature of lethal signalling in PDAC cells, and highlight the challenges and opportunities for activating cell death pathways through precision oncology treatments., (© 2021. Springer Nature Limited.)

Oncogenic KRAS mutation-driven pancreatic ductal adenocarcinoma is currently the fourth-leading cause of cancer-related deaths in the United States. Macroautophagy (hereafter "autophagy") is one of the lysosome-dependent degradation systems that can remove abnormal proteins, damaged organelles, or invading pathogens by activating dynamic membrane structures (e.g., phagophores, autophagosomes, and autolysosomes). Impaired autophagy (including excessive activation and defects) is a pathological feature of human diseases, including pancreatic cancer. However, dysfunctional autophagy has many types and plays a complex role in pancreatic tumor biology, depending on various factors, such as tumor stage, microenvironment, immunometabolic state, and death signals. As a modulator connecting various cellular events, pharmacological targeting of nonselective autophagy may lead to both good and bad therapeutic effects. In contrast, targeting selective autophagy could reduce potential side effects of the drugs used. In this review, we describe the advances and challenges of autophagy in the development and therapy of pancreatic cancer. Abbreviations : AMPK: AMP-activated protein kinase; CQ: chloroquine; csc: cancer stem cells; DAMP: danger/damage-associated molecular pattern; EMT: epithelial-mesenchymal transition; lncRNA: long noncoding RNA; MIR: microRNA; PanIN: pancreatic intraepithelial neoplasia; PDAC: pancreatic ductal adenocarcinoma; PtdIns3K: phosphatidylinositol 3-kinase; SNARE: soluble NSF attachment protein receptor; UPS: ubiquitin-proteasome system.

Dendritic cells (DCs) are antigen-presenting cells of the immune system, which play a key role in antitumor immunity by activating cytotoxic T cells. Here, we report that elevated ferroptosis, a lipid peroxidation-mediated cell death, impairs the maturation of DCs and their function in tumor suppression. Ferroptosis is selectively induced in DCs by the GXP4 inhibitor RSL3, but not the SLC7A11 inhibitor erastin. Ferroptotic DCs lose their ability to secrete pro-inflammatory cytokines (TNF and IL6) and express MHC class I in response to the maturation signal of lipopolysaccharide. Moreover, ferroptotic DCs fail to induce CD8 + T cells to produce IFNG/IFNγ. Mechanistically, PPARG/PPARγ, a nuclear receptor involved in the regulation of lipid metabolism, is responsible for RSL3-induced ferroptosis in DCs. Consequently, the genetic depletion of PPARG restores the maturation and function of DCs. Using immunogenic cell death-based DC vaccine models, we further demonstrate that PPARG-mediated ferroptosis of DCs limits antitumor immunity in mice. Together, these findings demonstrate a novel role of ferroptotic DCs in driving an immunosuppressive tumor microenvironment., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest or financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy with a 5-year survival rate below 10%. Its unique genetic makeup and tumor microenvironment produce a lack of response to current treatments, including chemotherapy, radiotherapy, and immunotherapy. Recent preclinical studies have revealed that ferroptosis, an iron-dependent form of nonapoptotic cell death driven by unrestricted lipid peroxidation, may be an attractive therapeutic goal in PDAC. Understanding the dual role of ferroptotic cell death in both promoting and suppressing tumor immunity, as well as its integrated regulatory mechanisms and signaling pathways, may lead to more effective treatment designs for clinical trials of PDAC and may minimize or delay the emergence of drug resistance or side effects., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a unique tumor microenvironment surrounded by an interlaced network of cancer and noncancerous cells. Recent works have revealed that the dynamic interaction between cancer cells and neuronal cells leads to perineural invasion (PNI), a clinical pathological feature of PDAC. The formation and function of PNI are dually regulated by molecular (e.g., involving neurotrophins, cytokines, chemokines, and neurotransmitters), metabolic (e.g., serine metabolism), and cellular mechanisms (e.g., involving Schwann cells, stromal cells, T cells, and macrophages). Such integrated mechanisms of PNI not only support tumor development, growth, invasion, and metastasis but also mediate the formation of pain, all of which are closely related to poor disease prognosis in PDAC. This review details the modulation, signaling pathways, detection, and clinical relevance of PNI and highlights the opportunities for further exploration that may benefit PDAC patients., (© 2021 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

Background: Aerobic glycolysis has a pivotal role in the carcinogenic process. The current understanding of the functional role and mechanism of UCHL3-related aerobic glycolysis in pancreatic cancer is far from comprehensive, therefore requires an in-depth analysis on this aspect., Methods: In the present research, the expressions of ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), lactate dehydrogenase A (LDHA) and Forkhead box protein M1 (FOXM1) were detected by qRT-PCR, Western blot and immunohistochemistry. The effects of UCHL3 knockdown or overexpression on pancreatic cancer cells were examined by determining cell viability and colony formation. Aerobic glycolysis was assessed according to glucose uptake, lactic acid production, and lactate dehydrogenase (LDH) activity. Dual-luciferase reporter assay was performed to detect LDHA promoter activity., Results: The results showed that UCHL3 expression was significantly increased in the pancreatic cancer tissues and cells, and that knocking down UCHL3 noticeably inhibited cell viability and aerobic glycolysis. Further investigations revealed that LDHA expression was promoted by UCHL3 and could be reduced by shFOXM1, and that low-expressed LDHA partly reversed the inhibition of aerobic glycolysis induced by overexpressed UCHL3., Conclusions: To conclude, this study demonstrates that UCHL3 plays a carcinogenic role by promoting aerobic glycolysis in pancreatic cancer, suggesting that UCHL3 may be a potential diagnostic and therapeutic target for the treatment of cancer.

Ferroptosis is a type of nonapoptotic cell death driven by lipid peroxidation. Here, we show a key role of MGST1 in inhibiting ferroptosis in cell cultures and mouse xenograft models. Ferroptosis activators induce MGST1 upregulation in human pancreatic ductal adenocarcinoma (PDAC) cell lines in an NFE2L2-dependent manner. The genetic depletion of MGST1 or NFE2L2 has a similar effect in promoting ferroptosis, whereas the re-expression of MGST1 restores the resistance of NFE2L2-knockdown cells to ferroptosis. MGST1 inhibits ferroptotic cancer cell death partly by binding to ALOX5, resulting in reduced lipid peroxidation. The expression of MGST1 is positively correlated with NFE2L2 expression in pancreatic tumors, which is implicated in the poor prognosis of patients with PDAC. These findings not only provide a valuable insight into the defense mechanism against ferroptotic cell death, but also indicate that targeting the MGST1 redox-sensitive pathway may be a promising strategy for the treatment of PDAC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: This study aimed to investigate the effect and mechanism of hypoxia on pancreatic cancer (PC) cell dedifferentiation and tumorigenic potential., Methods: Inhibition of hypoxia-inducible factor 1α (HIF-1α) and overexpression of Notch1 in PC HS766T cell lines were by lentiviral transfection. The expression of stem cell-specific markers C-X-C motif chemokine receptor 4, CD44, and Nestin was detected by immunofluorescence and Western blot assays. Cell invasion capacity was examined by Transwell assay. Tumorigenic potential was measured in an in situ tumor transplantation experiment. The expression of HIF-1α, Notch signals, and apoptosis signals was examined by Western blot assay., Results: Hypoxia promoted PC cells to dedifferentiate into stem-like cells by upregulating HIF-1α and activating Notch signals. Silencing of HIF-1α significantly repressed cell dedifferentiation and invasion, whereas overexpression of Notch1 reversed the effect of HIF-1α repression. In situ tumor transplantation experiment further confirmed that hypoxia promoted tumorigenic ability through upregulating HIF-1α. Moreover, the expression of HIF-1α and Notch1 was significantly increased in human PC tissues, and high expression of HIF-1α was correlated with poor survival rate., Conclusions: Hypoxia promoted PC cell dedifferentiation to stem-like cell phenotypes with high tumorigenic potential by activating HIF-1α/Notch signaling pathway, indicating a novel role in regulating PC progression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Adaptive immune resistance mediated by the cytokine interferon gamma (IFNG) still constitutes a major problem in cancer immunotherapy. We develop strategies for overcoming IFNG-mediated adaptive immune resistance in pancreatic ductal adenocarcinoma cancer (PDAC)., Design: We screened 429 kinase inhibitors for blocking IFNG-induced immune checkpoint (indoleamine 2,3-dioxygenase 1 (IDO1) and CD274) expression in a human PDAC cell line. We evaluated the ability of the cyclin-dependent kinase (CDK) inhibitor dinaciclib to block IFNG-induced IDO1 and CD274 expression in 24 human and mouse cancer cell lines as well as in primary cancer cells from patients with PDAC or ovarian carcinoma. We tested the effects of dinaciclib on IFNG-induced signal transducer and activator of transcription 1 activation and immunological cell death, and investigated the potential utility of dinaciclib in combination with IFNG for pancreatic cancer therapy in vivo, and compared gene expression levels between human cancer tissues with patient survival times using the Cancer Genome Atlas datasets., Results: Pharmacological (using dinaciclib) or genetic (using shRNA or siRNA) inactivation of CDK1/2/5 not only blocks JUN-dependent immune checkpoint expression, but also triggers histone-dependent immunogenic cell death in immortalised or primary cancer cells in response to IFNG. This dual mechanism turns an immunologically 'cold' tumour microenvironment into a 'hot' one, dramatically improving overall survival rates in mouse pancreatic tumour models (subcutaneous, orthotopic and transgenic models). The abnormal expression of CDK1/2/5 and IDO1 was associated with poor patient survival in several cancer types, including PDAC., Conclusion: CDK1/2/5 kinase activity is essential for IFNG-mediated cancer immunoevasion. CDK1/2/5 inhibition by dinaciclib provides a novel strategy to overcome IFNG-triggered acquired resistance in pancreatic tumour immunity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Although induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as an anticancer strategy, the metabolic basis of ferroptotic death remains poorly elucidated. Here, we show that glucose determines the sensitivity of human pancreatic ductal carcinoma cells to ferroptosis induced by pharmacologically inhibiting system xc - . Mechanistically, SLC2A1-mediated glucose uptake promotes glycolysis and, thus, facilitates pyruvate oxidation, fuels the tricyclic acid cycle, and stimulates fatty acid synthesis, which finally facilitates lipid peroxidation-dependent ferroptotic death. Screening of a small interfering RNA (siRNA) library targeting metabolic enzymes leads to identification of pyruvate dehydrogenase kinase 4 (PDK4) as the top gene responsible for ferroptosis resistance. PDK4 inhibits ferroptosis by blocking pyruvate dehydrogenase-dependent pyruvate oxidation. Inhibiting PDK4 enhances the anticancer activity of system xc - inhibitors in vitro and in suitable preclinical mouse models (e.g., a high-fat diet diabetes model). These findings reveal metabolic reprogramming as a potential target for overcoming ferroptosis resistance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ferroptosis is a type of iron-dependent regulated cell death, representing an emerging disease-modulatory mechanism. Transcription factors play multiple roles in ferroptosis, although the key regulator for ferroptosis in iron metabolism remains elusive. Using NanoString technology, we identify NUPR1, a stress-inducible transcription factor, as a driver of ferroptosis resistance. Mechanistically, NUPR1-mediated LCN2 expression blocks ferroptotic cell death through diminishing iron accumulation and subsequent oxidative damage. Consequently, LCN2 depletion mimics NUPR1 deficiency with respect to ferroptosis induction, whereas transfection-enforced re-expression of LCN2 restores resistance to ferroptosis in NUPR1-deficient cells. Pharmacological or genetic blockade of the NUPR1-LCN2 pathway (using NUPR1 shRNA, LCN2 shRNA, pancreas-specific Lcn2 conditional knockout mice, or the small molecule ZZW-115) increases the activity of the ferroptosis inducer erastin and worsens pancreatitis, in suitable mouse models. These findings suggest a link between NUPR1-regulated iron metabolism and ferroptosis susceptibility.

Pancreatic ductal adenocarcinoma (PDAC) is an immune resistant tumor. We recently demonstrated that inhibiting CDK1/2/5 by dinaciclib not only blocks immune checkpoint expression, but also triggers histone-dependent immunogenic cell death. This dual mechanism turns immunologically "cold" tumor microenvironment into a "hot" one, improves overall survival rates in mouse PDAC models., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Drug-induced ferroptosis, an iron-dependent regulatory necrosis, has been proposed for the therapy of pancreatic ductal adenocarcinoma. However, genetically engineered mouse models have revealed that high-iron diets or deletion of pancreatic GPX4 (a key repressor of ferroptosis) accelerate the development of mutant Kras -driven PDAC by activating the STING1/TMEM173-dependent DNA sensor pathway. Abbreviations ADM: acinar-to-ductal metaplasia; CGAS: cyclic GMP-AMP synthase; DAMP: damage-associated molecular pattern; GPX4: glutathione peroxidase 4; GEMM: genetically engineered mouse models; PDAC: pancreatic ductal adenocarcinoma; PanIN: pancreatic intraepithelial neoplasia, SLC7A11: solute carrier family 7 member 11; STING1: cGAMP-stimulator of interferon response cGAMP interactor 1; TME: tumor microenvironment; 8-OHG: 8-hydroxy-2'-deoxyguanosine., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel., Patients and Methods: Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines., Results: Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses ( P = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival ( P < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, P = 0.027, as well as increased immune cell tumor infiltration, P = 0.033). Overall survival ( P = 0.59) and relapse-free survival ( P = 0.55) did not differ between the two arms., Conclusions: The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity., (©2020 American Association for Cancer Research.)

Background: Circular RNAs (circRNAs) have recently been shown to play important roles in different tumors. However, their detailed roles and regulatory mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not well understood. This study aimed to identify enriched circRNAs and detect their functions and mechanisms in PDAC cells and tissues., Methods: circRNA-ASH2L (circ-ASH2L) was identified by circRNA microarray studies based on previous studies, and further detected in PDAC cells and samples by qRT-PCR. The functions of circ-ASH2L were identified by transwell, EdU, cell cycle or Tube formation assays. The regulatory mechanisms of circ-ASH2L were explored by WB, RIP, FISH, dual-luciferase assays, RNA pulldown or other assays., Results: We identified a circRNA (circ-ASH2L) based on our previous studies, detected its expression in different malignant cells and found that circ-ASH2L was highly expressed in pancreatic cells or tumor tissues and correlated with tumor malignancy. Further studies revealed that circ-ASH2L promoted tumor invasion, proliferation and angiogenesis by regulating miR-34a, thus regulate Notch 1 expression. Circ-ASH2L served as a miRNA sponge for miR-34a and promoted tumor progression in vivo. Finally, we analyzed circ-ASH2L expression in clinical tissues and found that high circ-ASH2L expression was correlated with lymphatic invasion and TNM stage and was an independent risk factor for pancreatic patient survival., Conclusions: circ-ASH2L play an important role in tumor invasion, and high circ-ASH2L may be a useful marker of PDAC diagnosis or progression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with poor prognosis, and the interaction between activated pancreatic stellate cells (PSCs) and PDAC cells plays an important role in the development of PDAC. The aim of this study was to identify gene changes in BXPC-3 after cross-talk with PSCs and reveal their potential mechanisms. The gene expression profiling analysis of BXPC-3 was completed after co-culture with primary PSCs for 48 h. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed, and the differentially expressed genes (DEGs) were identified by Agilent GeneSpring GX software. In total, 3657 DEGs were identified in BXPC-3, including 1881 up-regulated genes and 1776 downregulated genes. GO analysis results showed that upregulated DEGs were significantly enriched in biological processes (BP), including peptide metabolic process, response to stress and electron transport chain; the downregulated DEGs were significantly enriched in biological processes, including signaling, multicellular organism development and anatomical structure development. KEGG pathway analysis revealed that 19 pathways were upregulated and 32 pathways were downregulated, and that upregulated DEGs were enriched in protein export and glutathione metabolism, while the downregulated DEGs were enriched in axon guidance and focal adhesion. The top 10 upregulated genes and the top 10 downregulated genes were identified. By constructing PPI network, we selected out 10 key genes (TP53, SRC, IL6, JUN, ISG15, CAD, STAT1, OAS3, OAS1, VIM) and significant pathways. The associated survival analysis was performed and the SRC, IL-6, ISG15, STAT1, OAS3, OAS1 and VIM were proved to be related to worse overall survival time of PDAC patients. In conclusion, the present study indicated that the identified DEGs promote our understanding of the molecular mechanisms underlying the interaction between pancreatic cancer cells and PSCs and might be used as molecular targets in the future to study the role of tumor microenvironment in the progression of PDAC.

It is well known that as the king of cancer, pancreatic ductal adenocarcinoma (PDAC) has relatively malignant biological behavior and poor prognosis. The interaction between pancreatic stellate cells and PDAC cells promotes the development of PDAC. The aim of this study was to describe gene characteristics in pancreatic stellate cell (PSCs) after cross-talked with BXPC-3 and unravel their underlying mechanisms. The expression profiling analysis of genes in PSCs was completed after co-cultured with primary BXPC-3 for 48h. The Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis and gene ontology (GO) analysis were performed, and the differentially expressed genes (DEGs) were identified by Agilent GeneSpring GX program. In total, 1804 DEGs were filtered out in PSCs, including 958 up-regulated genes and 846 downregulated genes. GO analysis showed that the up-regulated DEGs were significantly enriched in biological processes (BP) such as defense response, immune system process and immune response; the down-regulated DEGs were significantly enriched in biological regulation and cytoskeleton organization. KEGG pathway analysis showed that 28 pathways were upregulated and 5 were downregulated. By constructing PPI network, we selected out 10 key genes (IL6，IL8, IL1B, BCL2, CCL2, CSF2, KIT, ICAM1, PTPRC and IGF1) and significant enriched pathways. In conclusion, the current study suggests that the filtered DEGs contribute to our understanding of the molecular mechanisms underlying the interaction between PSCs and pancreatic cancer cells, and might be used as molecular targets to further the study the role of tumor microenvironment in the progression of PDAC.

Background & Aims: Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice., Methods: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRas G12D/+ ;Tp53 R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;Kras G12D/+ mice crossed with Hmgb1 flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses., Results: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax -/- /Bak -/- cells), necroptosis (Ripk1 -/- , Ripk3 -/- , or Mlkl -/- cells), ferroptosis (Gpx4 -/- cells), or autophagy (Atg3 -/- , Atg5 -/- , Atg7 -/- , or Sqstm1 -/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34 + progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice., Conclusions: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Noninvasive detection of both early pancreatic neoplasia and metastases could enhance strategies to improve patient survival in this disease that is notorious for an extremely poor prognosis. There are almost no identifiable targets for non-invasive diagnosis by positron emission tomography (PET) for patients with pancreatic ductal adenocarcinoma (PDAC). Over-expression of the receptor for advanced glycation end products (RAGE) is found on the cell surface of both pre-neoplastic lesions and invasive PDAC. Here, a RAGE-specific single chain (scFv) was developed, specific for PET imaging in syngeneic mouse models of PDAC. An anti-RAGE scFv conjugated with a sulfo-Cy5 fluorescence molecule showed high affinity and selectivity for RAGE expressing pancreatic tumor cells and genetically engineered KRASG12D mouse models of PDAC. An in vivo biodistribution study was performed with the 64Cu-radiolabled scFv in a syngeneic murine pancreatic cancer model, demonstrating both the feasibility and potential of an anti-RAGE scFv for detection of PDAC. These studies hold great promise for translation into the clinic.

Chronic pancreatitis/pancreatic cancer (CP/PC) is characterized by fibrous connective tissue proliferation induced by activated pancreatic stellate cells (PSCs). Galectin-1 is upregulated in activated PSCs and is important for the continuing activation of PSCs. The aim of this study was to evaluate the effect of galectin-1 derived from activated PSCs on the progression of fibrosis in CP/PC. To this end, the expression of desmin, α-SMA, galectin-1, fibronectin and collagen type I in normal pancreatic, CP and PC tissues, as well as quiescent/activated PSCs, was investigated. The proliferation rate and migration ability of control, galectin-1-overexpressing and galectin-1-silenced PSCs were also evaluated, as well as the mRNA and protein expression of fibronectin, collagen type I, α-SMA, tissue inhibitors of metalloproteinases (TIMP)-1, MMP-2, Smad2 and TGF-β1. Furthermore, the effect of adding a TGF-β1 receptor inhibitor on the expression of these proteins was examined. The results revealed that the expression profile of desmin, α-SMA, galectin-1, fibronectin and collagen type I in the normal pancreas was similar to that of quiescent PSCs and the expression profile in CP/PC tissues was similar to that of activated PSCs. Furthermore, galectin-1-overexpressing PSCs exhibited a significantly higher proliferation rate and migration ability, while galectin-1-silenced PSCs exhibited a significantly lower proliferation rate and migration ability than the control PSCs. The expression of fibronectin, collagen type I, α-SMA, MMP-2 and TIMP-1 was also significantly higher in the galectin-1-overexpressing PSCs than the control PSCs and this effect was found to be mediated by the TGF-β1/Smad pathway. The trends in the expression of these factors were reversed in the galectin-1-silenced PSCs. From these findings, it can be concluded that overexpression of galectin-1 promotes PSC activity (proliferation and migration) and stimulates fibrosis by increasing extracellular matrix synthesis and decreasing the MMP/TIMP ratio via the TGF-β1/Smad pathway. Thus, galectin-1 may be a novel candidate for reversing or halting fibrosis progression in CP/PC.

Background & Aims: Induction of nonapoptotic cell death could be an approach to eliminate apoptosis-resistant tumors. We investigated necroptosis-based therapies in mouse models of pancreatic ductal adenocarcinoma cancer (PDAC)., Methods: We screened 273 commercially available kinase inhibitors for cytotoxicity against a human PDAC cell line (PANC1). We evaluated the ability of the aurora kinase inhibitor CCT137690 to stimulate necroptosis in PDAC cell lines (PANC1, PANC2.03, CFPAC1, MiaPaCa2, BxPc3, and PANC02) and the HEK293 cell line, measuring loss of plasma membrane integrity, gain in cell volume, swollen organelles, and cytoplasmic vacuoles. We tested the effects of CCT137690 in colon formation assays, and the effects of the necroptosis (necrostatin-1 and necrosulfonamide), apoptosis, autophagy, and ferroptosis inhibitors. We derived cells from tumors that developed in Pdx1-Cre;K-Ras G12D/+ ;p53 R172H/+ (KPC) mice. Genes encoding proteins in cell death pathways were knocked out, knocked down, or expressed from transgenes in PDAC cell lines. Athymic nude or B6 mice were given subcutaneous injections of PDAC cells or tail-vein injections of KPC tumor cells. Mice were given CCT137690 (80 mg/kg) or vehicle and tumor growth was monitored; tumor tissues were collected and analyzed by immunohistochemistry. We compared gene expression levels between human pancreatic cancer tissues (n = 130) with patient survival times using the online R2 genomics analysis and visualization platform., Results: CCT137690 induced necrosis-like death in PDAC cell lines and reduced colony formation; these effects required RIPK1, RIPK3, and MLKL, as well as inhibition of aurora kinase A (AURKA). AURKA interacted directly with RIPK1 and RIPK3 to reduce necrosome activation. AURKA-mediated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9 inhibited activation of the RIPK3 and MLKL necrosome. Mutations in AURKA (D274A) or GSK3β (S9A), or pharmacologic inhibitors of RIPK1 signaling via RIPK3 and MLKL, reduced the cytotoxic activity of CCT137690 in PDAC cells. Oral administration of CCT137690 induced necroptosis and immunogenic cell death in subcutaneous and orthotopic tumors in mice, and reduced tumor growth and tumor cell phosphorylation of AURKA and GSK3β. CCT137690 increased survival times of mice with orthotopic KPC PDACs and reduced tumor growth, stroma, and metastasis. Increased expression of AURKA and GSK3β mRNAs associated with shorter survival times of patients with pancreatic cancer., Conclusions: We identified the aurora kinase inhibitor CCT137690 as an agent that induces necrosis-like death in PDAC cells, via RIPK1, RIPK3, and MLKL. CCT137690 slowed growth of orthotopic tumors from PDAC cells in mice, and expression of AURKA and GSK3β associate with patient survival times. AURKA might be targeted for treatment of pancreatic cancer., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) driven by oncogenic K-Ras remains among the most lethal human cancers despite recent advances in modern medicine. The pathogenesis of PDAC is partly attributable to intrinsic chromosome instability and extrinsic inflammation activation. However, the molecular link between these two events in pancreatic tumorigenesis has not yet been fully established. Here, we show that intracellular high mobility group box 1 (HMGB1) remarkably suppresses oncogenic K-Ras-driven pancreatic tumorigenesis by inhibiting chromosome instability-mediated pro-inflammatory nucleosome release. Conditional genetic ablation of either single or both alleles of HMGB1 in the pancreas renders mice extremely sensitive to oncogenic K-Ras-driven initiation of precursor lesions at birth, including pancreatic intraepithelial neoplasms, intraductal papillary mucinous neoplasms, and mucinous cystic neoplasms. Loss of HMGB1 in the pancreas is associated with oxidative DNA damage and chromosomal instability characterized by chromosome rearrangements and telomere abnormalities. These lead to inflammatory nucleosome release and propagate K-Ras-driven pancreatic tumorigenesis. Extracellular nucleosomes promote interleukin 6 (IL-6) secretion by infiltrating macrophages/neutrophils and enhance oncogenic K-Ras signaling activation in pancreatic lesions. Neutralizing antibodies to IL-6 or histone H3 or knockout of the receptor for advanced glycation end products all limit K-Ras signaling activation, prevent cancer development and metastasis/invasion, and prolong animal survival in Pdx1-Cre;K-Ras G12D/+ ;Hmgb1 -/- mice. Pharmacological inhibition of HMGB1 loss by glycyrrhizin limits oncogenic K-Ras-driven tumorigenesis in mice under inflammatory conditions. Diminished nuclear and total cellular expression of HMGB1 in PDAC patients correlates with poor overall survival, supporting intracellular HMGB1 as a novel tumor suppressor with prognostic and therapeutic relevance in PDAC.

Ferroptosis is a form of regulated cell death driven by oxidative injury promoting lipid peroxidation, although detailed molecular regulators are largely unknown. Here, we show that heatshock 70-kDa protein 5 (HSPA5) negatively regulates ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, activating transcription factor 4 (ATF4) resulted in the induction of HSPA5, which in turn bound glutathione peroxidase 4 (GPX4) and protected against GPX4 protein degradation and subsequent lipid peroxidation. Importantly, the HSPA5-GPX4 pathway mediated ferroptosis resistance, limiting the anticancer activity of gemcitabine. Genetic or pharmacologic inhibition of the HSPA5-GPX4 pathway enhanced gemcitabine sensitivity by disinhibiting ferroptosis in vitro and in both subcutaneous and orthotopic animal models of PDAC. Collectively, these findings identify a novel role of HSPA5 in ferroptosis and suggest a potential therapeutic strategy for overcoming gemcitabine resistance. Cancer Res; 77(8); 2064-77. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Macroautophagy/autophagy is an evolutionarily conserved degradation pathway that maintains homeostasis. Ferroptosis, a novel form of regulated cell death, is characterized by a production of reactive oxygen species from accumulated iron and lipid peroxidation. However, the relationship between autophagy and ferroptosis at the genetic level remains unclear. Here, we demonstrated that autophagy contributes to ferroptosis by degradation of ferritin in fibroblasts and cancer cells. Knockout or knockdown of Atg5 (autophagy-related 5) and Atg7 limited erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation. Remarkably, NCOA4 (nuclear receptor coactivator 4) was a selective cargo receptor for the selective autophagic turnover of ferritin (namely ferritinophagy) in ferroptosis. Consistently, genetic inhibition of NCOA4 inhibited ferritin degradation and suppressed ferroptosis. In contrast, overexpression of NCOA4 increased ferritin degradation and promoted ferroptosis. These findings provide novel insight into the interplay between autophagy and regulated cell death.

Ferroptosis, a novel form of regulated cell death, is characterized by oxidative injury from iron accumulation and lipid peroxidation. In a natural product library screening for ferroptosis inhibitor, we found that baicalein is a potent inhibitor of erastin-induced ferroptosis in pancreatic cancer cells. Baicalein (also termed 5,6,7-trihydroxyflavone) is a flavonoid originally obtained from the roots of Scutellaria baicalensis and Scutellaria lateriflora. We showed that baicalein exhibits remarkable anti-ferroptosis activity compared with well-known ferroptosis inhibitors such as ferrostatin-1, liproxstatin-1, deferoxamine mesylate, and β-mercaptoethanol. At the biochemistry level, baicalein limits erastin-induced ferrous iron production, glutathione depletion, and lipid peroxidation. At the protein level, baicalein suppresses erastin-mediated degradation of glutathione peroxidase 4, a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. Thus, baicalein enhances cellular anti-ferroptosis capacity and could be a potential therapeutic agent for ferroptosis-associated tissue injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Galectin-1, a β-galactoside-binding protein implicated in cancer cell immune privilege, was highly expressed in activated pancreatic stellate cells (PSCs). This study was designed to investigate the relationship between PSC-derived galectin-1 and tumor immunity in pancreatic cancer. Isolated PSCs were identified as normal pancreas cells (hNPSCs) or pancreatic cancer cells (hCaPSCs) by immunohistochemical staining for α-SMA and vimentin, and galectin-1 expression was evaluated by Western blotting and quantitative RT-PCR. Apoptosis, caspase activity, and cytokine production (IL-6, IL-10, TNF-β, and IFN-γ) of T cells co-cultured with PSCs were evaluated, and immunohistochemical staining of galectin-1 was correlated with CD3 and clinicopathological variables in 66 pancreatic cancer and 10 normal pancreatic tissue samples. hCaPSCs exhibited higher galectin-1 expression than did hNPSCs, and hCaPSCs induced higher levels of apoptosis in T cells following co-culture. hCaPSCs activated caspase-9 and caspase-3 in the mitochondrial apoptotic pathway and stimulated secretion of Th2 cytokines (IL-6 and IL-10) but decreased secretion of Th1 cytokines (TNF-β and IFN-γ), compared with hNPSCs. Immunohistochemical staining indicated that galectin-1 and CD3 were more highly expressed in pancreatic cancer tissue. Galectin-1 expression was highest in poorly differentiated pancreatic cancer cells and lowest in well-differentiated pancreatic cancer cells and was associated with tumor size, lymph node metastasis, differentiation, and UICC stage. However, CD3 expression showed the opposite trend and was highest in well-differentiated pancreatic cancer cells and was associated with tumor differentiation and UICC stage. High expression of galectin-1 was associated with short survival, as was low expression of CD3. hCaPSC-derived galectin-1 enhanced apoptosis and anergy of T cells in pancreatic cancer, which contributes to the immune escape of pancreatic cancer cells.

Neutrophil extracellular traps (NETs) are formed when neutrophils expel their DNA, histones and intracellular proteins into the extracellular space or circulation. NET formation is dependent on autophagy and is mediated by citrullination of histones to allow for the unwinding and subsequent expulsion of DNA. NETs have an important role in the pathogenesis of several sterile inflammatory diseases, including malignancy, therefore we investigated the role of NETs in the setting of pancreatic ductal adenocarcinoma (PDA). Neutrophils isolated from two distinct animal models of PDA had an increased propensity to form NETs following stimulation with platelet activating factor (PAF). Serum DNA, a marker of circulating NET formation, was elevated in tumor bearing animals as well as in patients with PDA. Citrullinated histone H3 expression, a marker of NET formation, was observed in pancreatic tumors obtained from murine models and patients with PDA. Inhibition of autophagy with chloroquine or genetic ablation of receptor for advanced glycation end products (RAGE) resulted in decreased propensity for NET formation, decreased serum DNA and decreased citrullinated histone H3 expression in the pancreatic tumor microenvironment. We conclude that NETs are upregulated in pancreatic cancer through RAGE-dependent/autophagy mediated pathways.

Novel therapies need to be developed for patients with pancreatic cancer because of the poor outcomes of current regimens. Pancreatic cancer cells respond to the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C chemokine receptor type 7 (CXCR7)/C-X-C motif chemokine 12 (CXCL12)/high-mobility group box 1 signaling axis and this axis presents a novel target for therapy. C-X-C motif chemokine 12 stimulates CXCR4/CXCR7-bearing cells in a paracrine manner. C-X-C chemokine receptor type 4 and CXCR7 are transmembrane G protein-coupled receptors that, upon interaction with ligand CXCL12, activate downstream protein kinases that promote a more aggressive behavior. C-X-C chemokine receptor type 4 is expressed on most pancreatic cancer cells, whereas CXCR7 is primarily expressed on tumor-associated endothelium. High-mobility group box 1 promotes the CXCR4 and CXCL12 interaction, promoting angiogenesis and lymphangiogenesis. Hypoxia-inducible factor 1 is a potent stimulator of CXCR4 and CXCL12 expression, promoting more aggressive behavior. This receptor/ligand interaction can be disrupted by CXCR4 antagonists available and in clinical use to harvest bone marrow stem cells. Novel imaging strategies are now being developed at several centers to evaluate response to therapy and identify early recurrence. Thus, the CXCR4/CXCR7/CXCL12 interaction plays a critical role in cancer cell progression, proliferation, invasion, as well as metastasis and is a suitable target for therapy, imaging, as well as development of novel diagnostics.