Novel therapeutics to treat chronic pancreatitis: targeting pancreatic stellate cells and macrophages.

INTRODUCTION: Chronic pancreatitis (CP) is a persistent, recurrent, and progressive disorder that is characterized by chronic inflammation and irreversible fibrosis of the pancreas. It is associated with severe morbidity, resulting in intense abdominal pain, diabetes, exocrine and endocrine dysfunction, and an increased risk of pancreatic cancer. The etiological factors are diverse and the major risk factors include smoking, chronic alcoholism, as well as other environmental and genetic factors. The treatment and management of CP is challenging, and no definitive curative therapy is currently available. AREAS COVERED: This review paper aims to provide an overview of the different cell types in the pancreas that is known to mediate disease progression and outline potential novel therapeutic approaches and drug targets that may be effective in treating and managing CP. The information presented in this review was obtained by conducting a NCBI PubMed database search, using relevant keywords. EXPERT OPINION: In recent years, there has been an increased interest in the development of novel therapeutics for CP. A collaborative multi-disciplinary approach coupled with a consistent funding for research can expedite progress of translating the findings from bench to bedside.

Pancreatic stellate cells: Key players in pancreatic health and diseases (Review).

As a pluripotent cell, activated pancreatic stellate cells (PSCs) can differentiate into various pancreatic parenchymal cells and participate in the secretion of extracellular matrix and the repair of pancreatic damage. Additionally, PSCs characteristics allow them to contribute to pancreatic inflammation and carcinogenesis. Moreover, a detailed study of the pathogenesis of activated PSCs in pancreatic disease can offer promise for the development of innovative therapeutic strategies and improved patient prognoses. Therefore, the present study review aimed to examine the involvement of activated PSCs in pancreatic diseases and elucidate the underlying mechanisms to provide a viable therapeutic strategy for the management of pancreas­related diseases.

Proteomic profiling of small extracellular vesicles derived from mouse pancreatic cancer and stellate cells: Role in pancreatic cancer.

Small extracellular vesicles (sEVs) are cell-derived vesicles evolving as important elements involved in all stages of cancers. sEVs bear unique protein signatures that may serve as biomarkers. Pancreatic cancer (PC) records a very poor survival rate owing to its late diagnosis and several cancer cell-derived proteins have been reported as candidate biomarkers. However, given the pivotal role played by stellate cells (PSCs, which produce the collagenous stroma in PC), it is essential to also assess PSC-sEV cargo in biomarker discovery. Thus, this study aimed to isolate and characterise sEVs from mouse PC cells and PSCs cultured alone or as co-cultures and performed proteomic profiling and pathway analysis. Proteomics confirmed the enrichment of specific markers in the sEVs compared to their cells of origin as well as the proteins that are known to express in each of the culture types. Most importantly, for the first time it was revealed that PSC-sEVs are enriched in proteins (including G6PI, PGAM1, ENO1, ENO3, and LDHA) that mediate pathways related to development of diabetes, such as glucose metabolism and gluconeogenesis revealing a potential role of PSCs in pancreatic cancer-related diabetes (PCRD). PCRD is now considered a harbinger of PC and further research will enable to identify the role of these components in PCRD and may develop as novel candidate biomarkers of PC.

The nanobody targeting PD-L1 and CXCR4 counteracts pancreatic stellate cell-mediated tumour progression by disrupting tumour microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy worldwide owing to its complex tumour microenvironment and dense physical barriers. Stromal-derived factor-1 (SDF-1), which is abundantly secreted by tumour stromal cells, plays a pivotal role in promoting PDAC growth and metastasis. In this study, we investigated the impact and molecular mechanisms of the anti-PD-L1&CXCR4 bispecific nanobody on the TME and their consequent interference with PDAC progression. We found that blocking the SDF-1/CXCR4 signalling pathway delayed the epithelial-mesenchymal transition in pancreatic cancer cells. Anti-PD-L1&CXCR4 bispecific nanobody effectively suppress the secretion of SDF-1 by pancreatic stellate cells and downregulate the expression of smooth muscle actin alpha(α-SMA), thereby preventing the activation of cancer-associated fibroblasts by downregulating the PI3K/AKT signaling pathway. This improves the pancreatic tumour microenvironment, favouring the infiltration of T cells into the tumour tissue. In conclusion, our results suggest that the anti-PD-L1&CXCR4 bispecific nanobody exerts an antitumor immune response by changing the pancreatic tumour microenvironment. Hence, the anti-PD-L1&CXCR4 bispecific nanobody is a potential candidate for pancreatic cancer treatment.

Reactive Oxygen Species-Responsive Nanoparticles Toward Extracellular Matrix Normalization for Pancreatic Fibrosis Regression.

Pancreatic fibrosis (PF) is primarily characterized by aberrant production and degradation modes of extracellular matrix (ECM) components, resulting from the activation of pancreatic stellate cells (PSCs) and the pathological cross-linking of ECM mediated by lysyl oxidase (LOX) family members. The excessively deposited ECM increases matrix stiffness, and the over-accumulated reactive oxygen species (ROS) induces oxidative stress, which further stimulates the continuous activation of PSCs and advancing PF; challenging the strategy toward normalizing ECM homeostasis for the regression of PF. Herein, ROS-responsive and Vitamin A (VA) decorated micelles (named LR-SSVA) to reverse the imbalanced ECM homeostasis for ameliorating PF are designed and synthesized. Specifically, LR-SSVA selectively targets PSCs via VA, thereby effectively delivering siLOXL1 and resveratrol (RES) into the pancreas. The ROS-responsive released RES inhibits the overproduction of ECM by eliminating ROS and inactivating PSCs, meanwhile, the decreased expression of LOXL1 ameliorates the cross-linked collagen for easier degradation by collagenase which jointly normalizes ECM homeostasis and alleviates PF. This research shows that LR-SSVA is a safe and efficient ROS-response and PSC-targeted drug-delivery system for ECM normalization, which will propose an innovative and ideal platform for the reversal of PF.

Targeting ROCK2 improves macromolecular permeability in a 3D fibrotic pancreatic cancer microenvironment model.

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-ß (TGFß)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFß or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFß/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer.

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways.

BACKGROUND: Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. METHODS: ELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. RESULTS: ELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-ß1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. CONCLUSION: In conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Biodistribution and therapeutic efficacy of a gold nanoparticle-based targeted drug delivery system against pancreatic cancer.

Pancreatic cancer is characterized by desmoplasia; crosstalk between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) leads to the deposition of extracellular matrix proteins in the tumor environment resulting in poor vascularity. Targeting either PCCs or PSCs individually has produced mixed results, and there is currently no effective strategy to target both cell types simultaneously. Previously, we demonstrated, through in vitro cell culture experiments, that a specific gold nanoparticle-based nanoformulation containing the anti-EGFR antibody cetuximab (C225) as a targeting agent and gemcitabine as a chemotherapeutic agent effectively targets both PCCs and PSCs simultaneously. Herein, we extend our studies to test the ability of these in vitro tested nano formulations to inhibit tumor growth in an orthotopic co-implantation model of pancreatic cancer in vivo. Orthotopic tumors were established by co-implantation of equal numbers of PCCs and PSCs in the mouse pancreas. Among the various formulations tested, 5 nm gold nanoparticles coated with gemcitabine, cetuximab and poly-ethylene glycol (PEG) of molecular weight 1000 Da, which we named ACGP441000, demonstrated optimal efficacy in inhibiting tumor growth. The current study reveals an opportunity to target PCCs and PSCs simultaneously, by exploiting their overexpression of EGFR as a target, in order to inhibit pancreatic cancer growth.

Retinoic acid signaling pathway in pancreatic stellate cells: Insight into the anti-fibrotic effect and mechanism.

Pancreatic stellate cells (PSCs) are activated following loss of cytoplasmic vitamin A (retinol)-containing lipid droplets, which is a key event in the process of fibrogenesis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDCA). PSCs are the major source of cancer-associated fibroblasts (CAFs) that produce stroma to induce PDAC cancer cell growth, invasion, and metastasis. As an active metabolite of retinol, retinoic acid (RA) can regulate target gene expression in PSCs through its nuclear receptor complex (RAR/RXR or RXR/RXR) or transcriptional intermediary factor. Additionally, RA also has extranuclear and non-transcriptional effects. In vitro studies have shown that RA induces PSC deactivation which reduces extracellular matrix production through multiple modes of action, such as inhibiting TßRⅡ, PDGFRß, ß-catenin and Wnt production, downregulating ERK1/2 and JNK phosphorylation and suppressing active TGF-ß1 release. RA alone or in combination with other reagents have been demonstrated to have an effective anti-fibrotic effect on cerulein-induced mouse CP models in vivo studies. Clinical trial data have shown that repurposing all-trans retinoic acid (ATRA) as a stromal-targeting agent for human pancreatic cancer is safe and tolerable, suggesting the possibility of using RA for the treatment of CP and PDCA in humans. This review focuses on RA signaling pathways in PSCs and the effects and mechanisms of RA in PSC-mediated fibrogenesis as well as the anti-fibrotic and anti-tumor effects of RA targeting PSCs or CAFs in vitro and in vivo, highlighting the potential therapies of RA against CP and PDAC.

Pirfenidone alleviates chronic pancreatitis via suppressing the activation of pancreatic stellate cells and the M1 polarization of macrophages.

In the realm of fibroinflammatory conditions, chronic pancreatitis (CP) stands out as a particularly challenging ailment, lacking a dedicated, approved treatment. The potential of Pirfenidone (PFD), a drug originally used for treating idiopathic pulmonary fibrosis (IPF), in addressing CP's fibrotic aspects has sparked new interest. This investigation focused on the role of PFD in diminishing fibrosis and immune response in CP, using a mouse model induced by caerulein. The research extended to in vitro studies examining the influence of PFD on pancreatic stellate cells' (PSCs) behavior and the polarization of macrophages into M1 and M2 types. Advanced techniques like RNA sequencing and comprehensive data analyses were employed to decode the molecular interactions of PFD with PSCs. Supplementary experiments using techniques such as quantitative real-time PCR, western blotting, and immunofluorescence were also implemented. Results showed a notable reduction in pancreatic damage in PFD-treated mice, manifested through decreased acinar cell atrophy, lower collagen deposition, and a reduction in macrophage presence. Further investigation revealed PFD's capacity to hinder PSCs' migration, growth, and activation, alongside a reduction in the production and secretion of extracellular matrix proteins. This effect is primarily achieved by interfering with signaling pathways such as TGF-ß/Smad, Wnt/ß-catenin, and JAK/STAT. Additionally, PFD selectively hampers M1 macrophage polarization through the STAT3 pathway, without impacting M2 polarization. These outcomes highlight PFD's dual mechanism in moderating PSC activity and M1 macrophage polarization, positioning it as a promising candidate for CP therapy.

Disruption of Super-Enhancers in Activated Pancreatic Stellate Cells Facilitates Chemotherapy and Immunotherapy in Pancreatic Cancer.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers.

Chronic exposure to BDE-47 aggravates acute pancreatitis and chronic pancreatitis by promoting acinar cell apoptosis and inflammation.

The effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a persistent environmental pollutant commonly used as a flame retardant in various consumer products, on pancreatitis has not been clearly elucidated, although it has been reported to be toxic to the liver, nervous system, and reproductive system. Acute pancreatitis (AP) and chronic pancreatitis (CP) models were induced in this study by intraperitoneal injection of caerulein. The aim was to investigate the impact of BDE-47 on pancreatitis by exposing the animals to acute (1 week) or chronic (8 weeks) doses of BDE-47 (30 mg/kg in the low-concentration group and 100 mg/kg in the high-concentration group). Additionally, BDE-47 was utilized to stimulate mouse bone marrow-derived macrophages, pancreatic primary stellate cells, and acinar cells in order to investigate the impact of BDE-47 on pancreatitis. In vivo experiments conducted on mice revealed that chronic exposure to BDE-47, rather than acute exposure, exacerbated the histopathological damage of AP and CP, leading to elevated fibrosis in pancreatic tissue and increased infiltration of inflammatory cells in the pancreas. In vitro experiments showed that BDE-47 can promote the expression of the inflammatory cytokines Tnf-α and Il-6 in M1 macrophages, as well as promote acinar cell apoptosis through the activation of the PERK and JNK pathways via endoplasmic reticulum stress. The findings of this study imply chronic exposure to BDE-47 may exacerbate the progression of both AP and CP by inducing acinar cell apoptosis and dysregulating inflammatory responses.

Mouse islet-derived stellate cells are similar to, but distinct from, mesenchymal stromal cells and influence the beta cell function.

AIMS: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes-related conditions. We have identified a novel population of stromal cells within islets of Langerhans - islet stellate cells (ISCs) - which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. METHODS: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell-surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co-culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. RESULTS: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co-culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co-culture with MSCs. CONCLUSIONS: ISCs are a specific sub-type of islet-derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes.

Therapeutic Strategies for Pancreatic-Cancer-Related Type 2 Diabetes Centered around Natural Products.

Pancreatic ductal adenocarcinoma (PDAC), a highly malignant neoplasm, is classified as one of the most severe and devastating types of cancer. PDAC is a notable malignancy that exhibits a discouraging prognosis and a rising occurrence. The interplay between diabetes and pancreatic cancer exhibits a reciprocal causation. The identified metabolic disorder has been observed to possess noteworthy consequences on health outcomes, resulting in elevated rates of morbidity. The principal mechanisms involve the suppression of the immune system, the activation of pancreatic stellate cells (PSCs), and the onset of systemic metabolic disease caused by dysfunction of the islets. From this point forward, it is important to recognize that pancreatic-cancer-related diabetes (PCRD) has the ability to increase the likelihood of developing pancreatic cancer. This highlights the complex relationship that exists between these two physiological states. Therefore, we investigated into the complex domain of PSCs, elucidating their intricate signaling pathways and the profound influence of chemokines on their behavior and final outcome. In order to surmount the obstacle of drug resistance and eliminate PDAC, researchers have undertaken extensive efforts to explore and cultivate novel natural compounds of the next generation. Additional investigation is necessary in order to comprehensively comprehend the effect of PCRD-mediated apoptosis on the progression and onset of PDAC through the utilization of natural compounds. This study aims to examine the potential anticancer properties of natural compounds in individuals with diabetes who are undergoing chemotherapy, targeted therapy, or immunotherapy. It is anticipated that these compounds will exhibit increased potency and possess enhanced pharmacological benefits. According to our research findings, it is indicated that naturally derived chemical compounds hold potential in the development of PDAC therapies that are both safe and efficacious.

Arachidonate 15-lipoxygenase-mediated production of Resolvin D5n-3 DPA abrogates pancreatic stellate cell-induced cancer cell invasion.

Activation of pancreatic stellate cells (PSCs) to cancer-associated fibroblasts (CAFs) is responsible for the extensive desmoplastic reaction observed in PDAC stroma: a key driver of pancreatic ductal adenocarcinoma (PDAC) chemoresistance leading to poor prognosis. Specialized pro-resolving mediators (SPMs) are prime modulators of inflammation and its resolution, traditionally thought to be produced by immune cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling PSCs as well as primary human CAFs express enzymes and receptors to produce and respond to SPMs. Human PSC/CAF SPM secretion profile can be modulated by rendering these cells activated [transforming growth factor beta (TGF-ß)] or quiescent [all-trans retinoic acid (ATRA)]. ATRA-induced nuclear translocation of arachidonate-15-lipoxygenase (ALOX15) was linked to increased production of n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA), among other SPMs. Inhibition of RvD5n-3 DPA formation increases cancer cell invasion, whereas addback of this molecule reduced activated PSC-mediated cancer cell invasion. We also observed that circulating concentrations of RvD5n-3 DPA levels were decreased in peripheral blood of metastatic PDAC patients when compared with those measured in plasma of non-metastatic PDAC patients. Together, these findings indicate that RvD5n-3 DPA may regulate cancer-stroma cross-talk and invasion.

The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and - 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Pancreatic stellate cell-derived exosomal tRF-19-PNR8YPJZ promotes proliferation and mobility of pancreatic cancer through AXIN2.

The pancreatic stellate cells (PSCs) play an important role in the development of pancreatic cancer (PC) through mechanisms that remain unclear. Exosomes secreted from PSCs act as mediators for communication in PC. This study aimed to explore the role of PSC-derived exosomal small RNAs derived from tRNAs (tDRs) in PC cells. Exosomes from PSCs were extracted and used to detect their effects on PC cell proliferation, migration and invasion. Exosomal tDRs profiling was performed to identify PSC-derived exosomal tDRs. ISH and qRT-PCR were used to examine the tRF-19-PNR8YPJZ levels and clinical value in clinical samples. The biological function of exosomal tRF-19-PNR8YPJZ was determined using the CCK-8, clone formation, wound healing and transwell assays, subcutaneous tumour formation and lung metastatic models. The relationship between the selected exosomal tRF-19-PNR8YPJZ and AXIN2 was determined by RNA sequencing, luciferase reporter assay. PSC-derived exosomes promoted the proliferation, migration, and invasion of PC cells. Novel and abundant tDRs are found to be differentially expressed in PANC-1 cells after treatment with PSC-derived exosomes, such as tRF-19-PNR8YPJZ. PC tissue samples showed markedly higher levels of tRF-19-PNR8YPJZ than normal controls. Patients with PC exhibiting high tRF-19-PNR8YPJZ expression had a highly lymph node invasion, metastasis, perineural invasion, advanced clinical stage and poor overall survival. Exosomal tRF-19-PNR8YPJZ from PSCs targeted AXIN2 in PC cells and decreased its expression, thus activating the Wnt pathway and promoting proliferation and metastasis. Exosomal tRF-19-PNR8YPJZ from PSCs promoted proliferation and metastasis in PC cells via AXIN2.

Differential location of growth factors and pancreatic stellate cell activation in chronic pancreatitis.

Pancreatic fibrosis is characterized by the activation of pancreatic stellate cells leading to the expression of smooth muscle actin (α-SMA). Normal pancreatic tissue has predominantly quiescent stellate cells in periductal and perivascular locations, which do not express α-SMA. We aimed at studying the immunohistochemistry (IHC) expression pattern of α-SMA, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-ß) in the resected specimen of chronic pancreatitis. Twenty biopsies from resected specimens of patients with chronic pancreatitis were included. The expression was measured in comparison to positive control biopsies (breast carcinoma for PDGF-BB and TGF-ß and appendicular tissue for α-SMA) and scored based on a semi-quantitative system based on staining intensity. The percentage of positive cells was used for objective scoring, which ranged from 0 to 15. The scoring was done separately for acini, ducts, stroma and islet cell. All patients had undergone surgery for refractory pain and the median duration of symptoms was 48 months. On IHC, α-SMA was not expressed in the acini, ducts or islets, but had high expression in the stromal regions (vs. acini, ducts and islet, p < 0.05), TGF-ß1 was also expressed maximally in islet cells; however, the distribution among all locations was statistically similar. α-SMA expression in the pancreatic stroma is an indicator of the concentration of activated stellate cells in the stroma, a site for genesis of fibrosis under the influence of growth factors in the local milieu.

Reg family proteins contribute to inflammation and pancreatic stellate cells activation in chronic pancreatitis.

Chronic pancreatitis (CP) is a disease characterized by the inflammation and destruction of pancreatic tissue, leading to the replacement of functional tissue with fibrotic tissue. The regenerating gene (Reg) family proteins have recently been implicated in the repair and regeneration of inflamed pancreatic tissue, though the exact mechanisms of their involvement in the pathogenesis of CP are not yet fully understood. To investigate the role of Reg family proteins in CP, we generated global knockout mice (Reg-/-) for Reg1-3 (Reg1,2,3a,3b,3d,3g) genes using the CRISPR/Cas9 system. We then investigated the effect of Reg family protein deficiency in a genetic model of CP (X-SPINK1) mice by knocking out Reg1-3 genes. We examined pancreatic morphology, inflammatory cytokines expression, and activation of pancreatic stellate cells (PSCs) at different ages. Reg-/- mice showed no abnormalities in general growth and pancreas development. Deficiency of Reg1-3 in CP mice led to a reduction in pancreatic parenchymal loss, decreased deposition of collagen, and reduced expression of proinflammatory cytokines. Additionally, Reg proteins were found to stimulate PSCs activation. Overall, our study suggests that Reg1-3 deficiency can lead to the remission of CP and Reg family proteins could be a potential therapeutic target for the treatment of CP.

PITX2 in pancreatic stellate cells promotes EMT in pancreatic cancer cells via the Wnt/ß-catenin pathway.

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/ß-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/ß-catenin signaling.