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.

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.

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.

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.

5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFß1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFß1 treatment exerted a more substantial effect than TGFß1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

Synergistic autophagy blockade and VDR signaling activation enhance stellate cell reprogramming in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic tumor microenvironment (TME) consisting of abundant activated pancreatic stellate cells (PSCs). PSCs play a key role in the refractory responses of PDAC to immunotherapy and chemotherapy and deactivating PSCs into quiescence through vitamin D receptor (VDR) signaling activation is a promising strategy for PDAC treatment. We observed p62 loss in PSCs hindered the deactivation efficacy of VDR ligands, and hypothesized that reversing p62 levels by inhibiting autophagy processing, which is responsible for p62 loss, could sensitize PSCs toward VDR ligands. Herein, we constructed a PSC deactivator with dual functions of VDR activation and autophagy inhibition, utilizing a pH-buffering micelle (LBM) with an inherent ability to block autophagic flux to encapsulate calcipotriol (Cal), a VDR ligand. This Cal-loaded LBM (C-LBM) could efficiently reprogram PSCs, modulate the fibrotic TME, and alter immunosuppression. In combination with PD-1 antagonists and chemotherapy, C-LBM showed superior antitumor efficacy and significantly prolonged the survival of PDAC mice. These findings suggest that synergistic autophagy blockade and VDR signaling activation are promising therapeutic approaches to reprogram PSCs and improve the PDAC response to immunotherapy.

Cold Atmospheric Plasma Does Not Affect Stellate Cells Phenotype in Pancreatic Cancer Tissue in Ovo.

Pancreatic ductal adenocarcinoma (PDAC) is a challenging neoplastic disease, mainly due to the development of resistance to radio- and chemotherapy. Cold atmospheric plasma (CAP) is an alternative technology that can eliminate cancer cells through oxidative damage, as shown in vitro, in ovo, and in vivo. However, how CAP affects the pancreatic stellate cells (PSCs), key players in the invasion and metastasis of PDAC, is poorly understood. This study aims to determine the effect of an anti-PDAC CAP treatment on PSCs tissue developed in ovo using mono- and co-cultures of RLT-PSC (PSCs) and Mia PaCa-2 cells (PDAC). We measured tissue reduction upon CAP treatment and mRNA expression of PSC activation markers and extracellular matrix (ECM) remodelling factors via qRT-PCR. Protein expression of selected markers was confirmed via immunohistochemistry. CAP inhibited growth in Mia PaCa-2 and co-cultured tissue, but its effectiveness was reduced in the latter, which correlates with reduced ki67 levels. CAP did not alter the mRNA expression of PSC activation and ECM remodelling markers. No changes in MMP2 and MMP9 expression were observed in RLT-PSCs, but small changes were observed in Mia PaCa-2 cells. Our findings support the ability of CAP to eliminate PDAC cells, without altering the PSCs.

ESE3/EHF, a promising target of rosiglitazone, suppresses pancreatic cancer stemness by downregulating CXCR4.

BACKGROUND AND AIMS: The crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor (EHF) as a key molecule in decreasing the sensitivity of pancreatic cancer (PC) cells to CSCs' niche stimulus. We also explored a therapeutic strategy to restore the expression of EHF. DESIGN: We used a LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse model and samples from patients with PC. Immunostaining, flow cytometry, sphere formation assays, anchorage-independent growth assay, in vivo tumourigenicity, reverse transcription PCR, chromatin immunoprecipitation (ChIP) and luciferase analyses were conducted in this study. RESULTS: CXCL12 derived from pancreatic stellate cells (PSCs) mediates the crosstalk between PC cells and PSCs to promote PC stemness. Tumorous EHF suppressed CSC stemness by decreasing the sensitivity of PC to CXCL12 stimulus and inhibiting the crosstalk between PC and CSC-supportive niches. Mechanically, EHF suppressed the transcription of the CXCL12 receptor CXCR4. EHF had a cell autonomous role in suppressing cancer stemness by inhibiting the transcription of Sox9, Sox2, Oct4 and Nanog. Rosiglitazone suppressed PC stemness and inhibited the crosstalk between PC and PSCs by upregulating EHF. Preclinical KPC mouse cohorts demonstrated that rosiglitazone sensitised PDAC to gemcitabine therapy. CONCLUSIONS: EHF decreased the sensitivity of PC to the stimulus from PSC-derived CSC-supportive niche by negatively regulating tumorous CXCR4. Rosiglitazone could be used to target PC stem cells and the crosstalk between CSCs and their niche by upregulating EHF.

TGF-ß1 induced proliferation, migration, and ECM accumulation through the SNHG11/miR-34b/LIF pathway in human pancreatic stellate cells.

Chronic pancreatitis (CP) is a chronic inflammatory and fibrotic disease of the pancreas, and activated pancreatic stellate cells (PSCs) play a vital role in the progression of pancreatic fibrosis in CP. It has been reported that long non-coding RNA small nucleolar RNA host gene 11 (SNHG11) is highly expressed in chronic pancreatitis (CP) patients. However, the role of SNHG11 in CP progression is unclear. The purport of the study was to survey the role of SNHG11 in CP. We employed transforming growth factor (TGF)-beta1 (TGF-ß1) to activate human pancreatic stellate cells (PSCs). Expression of SNHG11 was assessed with qRT-PCR. Loss-of-function experiments were executed to evaluate the effects of SNHG11 on the proliferation and migration of TGF-ß1-treated PSCs. Some protein levels were detected by western blotting. The regulatory mechanism of SNHG11 was verified by the dual-luciferase reporter and RIP assays. As a result, SNHG11 was upregulated in plasma of CP patients and TGF-ß1-treated PSCs. Also, SNHG11 inhibition reduced TGF-ß1-induced proliferation, migration, and ECM accumulation in PSCs. Mechanistically, SNHG11 regulated leukemia inhibitory factor (LIF) expression by sponging miR-34b. Furthermore, miR-34b inhibitor abolished SNHG11 silencing-mediated effects on TGF-ß1-treated PSC proliferation, migration, and ECM accumulation. LIF overexpression counteracted the repressive influence of miR-34b mimic on proliferation, migration, and ECM accumulation of TGF-ß1-treated PSCs. In conclusion, SNHG11 knockdown reduced TGF-ß1-induced PSC proliferation, migration, and ECM accumulation by the miR-34b/LIF axis.

Melatonin Induces Apoptosis and Modulates Cyclin Expression and MAPK Phosphorylation in Pancreatic Stellate Cells Subjected to Hypoxia.

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM-1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.

Nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats.

AIM: Smoking has been considered as a risk factor of chronic pancreatitis (CP), but the potential mechanism is still unknown. The major pathological feature of CP is pancreatic fibrosis, whose major functional cells are pancreatic stellate cells (PSCs). Nicotine is the major component of cigarette smoke, our recent study suggested that nicotine has the potential to facilitate pancreatic fibrosis in CP. This study was aimed to analyze the function and mechanism of nicotine on PSCs and pancreatic fibrosis in rats. MATERIALS AND METHODS: In vivo, a rat CP model was induced by intraperitoneal injection of 20 % L-arginine hydrochloride (200 mg/100 g) at 1 h intervals twice per week, nicotine was injected subcutaneously at a dose of 1 mg/kg body weight per day. After four weeks, the pancreatic tissue was collected for H&E, Masson and immunohistochemical staining. In vitro, primary rPSCs were isolated from rats and treated with nicotine (0.1 µM and 1 µM). The proliferation、apoptosis、α-SMA expression、extracellular matrix (ECM) metabolism and α7nAChR-mediated JAK2/STAT3 signaling pathway of rPSCs were detected by CCK-8 assay、flow cytometry、real-time Q-PCR and western blotting analysis. The α7nAChR antagonist α-bungarotoxin (α-BTX) was used to perform inhibition experiments. KEY FINDINGS: Nicotine increased pancreatic damage, collagen deposition and activation of PSCs in the CP rat model. In rPSCs, the proliferation, α-SMA expression and ECM formation were significantly promoted by nicotine in a dose-dependent manner. Meanwhile, the apoptosis of rPSCs was significantly reduced after nicotine treatment. Moreover, nicotine also activated the α7nAChR-mediated JAK2/STAT3 signaling pathway in rPSCs. These effects of nicotine on rPSCs were blocked by α-BTX. SIGNIFICANCE: Our finding in this research suggests that nicotine facilitates pancreatic fibrosis by promoting activation of pancreatic stellate cells via α7nAChR-mediated JAK2/STAT3 signaling pathway in rats, partly revealing the mechanism of smoking on chronic pancreatitis.

Thalidomide Reduces Activation of Murine Pancreatic Stellate Cells by Inhibiting the TGF-ß/Smad Pathway.

OBJECTIVE: The aim of this study was to investigate the effects and mechanism of thalidomide on pancreatic stellate cell (PSC) activation in mice and to find the optimal timing of thalidomide administration. METHODS: PSCs, isolated from mouse pancreas tissue, were divided into five groups with specific treatments: (A) control PSCs (PSC), (B) PSCs induced by TGF-ß1 (PSC+TGF-ß1), (C) PSCs induced by TGF-ß1 followed by thalidomide (PSC+TGF-ß1+Thalidomide), (D) PSCs receiving TGF-ß1 and thalidomide simultaneously (PSC+(TGF-ß1+Thalidomide)), and (E) PSCs treated with thalidomide only (PSC+Thalidomide). We measured the effects of thalidomide on PSC activation by detecting the expression of α-SMA, collagen type I, and the TGF-ß/Smad pathway through quantitative real-time PCR and Western blot analysis. RESULTS: Compared with TGF-ß1 alone, thalidomide significantly inhibited PSC activation by reducing α-SMA expression (P<0.05) and decreasing collagen type I deposition (P<0.05). PSCs treated with thalidomide alone showed lower expression of α-SMA and collagen type I than those treated with thalidomide and TGF-ß1 at random order (P<0.01). Thalidomide downregulated TGF-ß1 and Smad3 and upregulated Smad7 (P<0.05). CONCLUSION: Thalidomide could repress PSC activation and alleviate fibrosis by regulating the TGF-ß/Smad pathway. Preventive use of thalidomide had maximum effect, and there was no evidence for the reversal of the activation of quiescent PSCs.

Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

Interleukin-6 participates in human pancreatic stellate cell activation and collagen I production via TGF-ß1/Smad pathway.

Pancreatic stellate cells (PSCs) play a key role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-ß1 (TGF-ß1) is a major regulator of PSC activation and extracellular matrix production. Interleukin-6 (IL-6) has shown to participate in TGF-ß1 production and rat PSC activation. This study aimed to investigate whether IL-6 promotes human PSC activation and collagen 1(Col1) production through the TGF-ß1/Smad pathway. Our results showed that the expression of IL-6 and IL-6R in activated PSCs and macrophages (Mφs) were enhanced in the pancreas of ACP compared to healthy controls and that the mRNA expression of IL-6, IL-6R, TGF-ß1, α-SMA or Col1a1 were significantly increased in the pancreas of ACP, showing positive correlations between elevated IL-6 levels and either TGF-ß1 or α-SMA or Col1a1 levels and between elevated TGF-ß1 levels and α-SMA or Col1a1 levels. In in vitro studies, we identified that IL-6R expression or IL-6 and TGF-ß1 secretions were significantly increased in, respectively, Mφs and PSCs by ethanol (EtOH) or lipopolysaccharide (LPS) stimulation while EtOH- or LPS-induced α-SMA or Col1a1 mRNA and protein production in PSCs were partially blocked by IL-6 antibody. IL-6-induced TGF-ß1 production in PSCs was antagonized by si-IL-6R RNA or by an inhibitor of STAT3. Additionally, IL-6-promoted α-SMA or Col1a1 protein production was blocked by TGF-ß1 antibody and IL-6-induced phosphorylation of Smad2/3 and transcription of α-SMA and Col1a1 mRNA were antagonized by si-TGF-ß1 RNA. Our findings indicate that IL-6 contributes to PSC activation and Col1 production through up-regulation of TGF-ß1/Smad2/3 pathway.

Silencing PCBP2 normalizes desmoplastic stroma and improves the antitumor activity of chemotherapy in pancreatic cancer.

Rationale: Dense desmoplastic stroma is a fundamental characteristic of pancreatic ductal adenocarcinoma (PDAC) and comprises up to 80% of the tumor mass. Type I collagen is the major component of the extracellular matrix (ECM), which acts as a barrier to impede the delivery of drugs into the tumor microenvironment. While the strategy to deplete PDAC stroma has failed in clinical trials, normalization of the stroma to allow chemotherapy to kill the tumor cells in the "nest" could be a promising strategy for PDAC therapy. We hypothesize that silencing the poly(rC)-binding protein 2 (αCP2, encoded by the PCBP2 gene) leads to the destabilization and normalization of type I collagen in the PDAC stroma. Methods: We develop a micro-flow mixing method to fabricate a peptide-based core-stabilized PCBP2 siRNA nanocomplex to reverse the accumulation of type I collagen in PDAC tumor stroma. Various in vitro studies were performed to evaluate the silencing activity, cellular uptake, serum stability, and tumor penetration of the PCBP2 siRNA nanocomplex. We also investigated the penetration of small molecules in stroma-rich pancreatic cancer spheroids after the treatment with the PCBP2 siRNA nanocomplex. The anti-tumor activity of the PCBP2 siRNA nanocomplex and its combination with gemcitabine was evaluated in an orthotopic stroma-rich pancreatic cancer mouse model. Results: Silencing the PCBP2 gene using siRNA reverses the accumulation of type I collagen in human pancreatic stellate cells (PSCs) and mouse NIH 3T3 fibroblast cells. The siRNA nanocomplex significantly reduces ECM production and enhances drug penetration through desmoplastic tumor stroma. The combination of gemcitabine with the PCBP2 siRNA nanocomplex markedly suppresses the tumor progression in a desmoplastic PDAC orthotopic mouse model. Conclusion: This approach provides a new therapeutic avenue to improve the antitumor efficacy of PDAC therapies by normalizing tumor stroma using the PCBP2 siRNA nanocomplex.

Regulation of p38 MAPK and glucocorticoid receptor activation by hydrocortisone in mono-and co-cultured pancreatic acinar and stellate cells.

BACKGROUND/OBJECTIVES: Acute pancreatitis develops as an inflammatory response to pancreatic tissue injury. Postoperative pancreatitis has recently been associated with increased occurrence of complications. Activation of the mitogen-activated protein kinase p38 (p38 MAPK) pathway occurs early in acute pancreatitis and its inhibition has been suggested to alleviate pancreatic inflammation. Glucocorticoids are potent anti-inflammatory steroids whose use in the management of acute pancreatitis remains controversial. Our aim was to examine the effect of crosstalk between pancreatic acinar cells (PACs) and stellate cells (PSCs) on p38 MAPK and glucocorticoid receptor (GR) activation and to assess the impact of hydrocortisone on these events. METHODS: The long-term co-culture setting for mouse PACs and PSCs developed in our laboratory was used. Parallel 4d mono- and co-cultures with or without 10 nM hydrocortisone were performed followed by immunocytochemical analysis of nuclear GR and phospho-p38 MAPK (pp38 MAPK). RESULTS: Hydrocortisone inhibited pp38 MAPK up-regulation evoked by co-culture in PACs and PSCs and increased nuclear translocation of GR in PAC monocultures and in co-cultured PACs and PSCs. In PSC monocultures and co-cultured PACs, ligand-independent expression of nuclear GR was observed. In the former no change in nuclear GR but a significant decrease in total GR as analyzed by Western blot was caused by hydrocortisone. CONCLUSIONS: Cellular microenvironment plays a significant role on p38 MAPK and GR activation in PACs and PSCs. Hydrocortisone is an effective means to inhibit p38 MAPK activation in PACs and PSCs. Both ligand-dependent and -independent regulatory roles for GR are suggested in the exocrine pancreas.

Heat Shock Protein-90 Inhibition Alters Activation of Pancreatic Stellate Cells and Enhances the Efficacy of PD-1 Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC), or cancer-associated fibroblasts (CAF). Targeting inflammatory pathways present within the stroma may improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90) is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC/CAF has not been explored in detail. We hypothesized that Hsp90 inhibition would limit inflammatory signals, thereby reprogramming the PDAC tumor microenvironment to enhance sensitivity to PD-1 blockade. Treatment of immortalized and primary patient PSC/CAF with the Hsp90 inhibitor XL888 decreased IL6, a key cytokine that orchestrates immune changes in PDAC at the transcript and protein level in vitro XL888 directly limited PSC/CAF growth and reduced Jak/STAT and MAPK signaling intermediates and alpha-SMA expression as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors. Tumors from mice treated with both XL888 and anti-PD-1 had a significantly increased CD8+ and CD4+ T-cell infiltrate and a unique transcriptional profile characterized by upregulation of genes associated with immune response and chemotaxis. These data demonstrate that Hsp90 inhibition directly affects PSC/CAF in vitro and enhances the efficacy of anti-PD-1 blockade in vivo.

FAK inhibition radiosensitizes pancreatic ductal adenocarcinoma cells in vitro.

INTRODUCTION: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase protein frequently overexpressed in cancer and has been linked to an increase in the stem cell population of tumors, resistance to therapy, and metastatic spread. Pharmacological FAK inhibition in pancreatic cancer has received increased attention over the last few years, either alone or in combination with other therapeutics including chemotherapy and immunotherapy. However, its prognostic value and its role in radioresistance of pancreatic ducal adenocarcinoma (PDAC) is unknown. METHODS AND MATERIALS: Using the TCGA and GTEx databases, we investigated the genetic alterations and mRNA expression levels of PTK2 (the encoding-gene for FAK) in normal pancreatic tissue and pancreatic cancer and its impact on patient survival. Furthermore, we evaluated the expression of FAK and its tyrosine domain Ty-397 in three pancreatic cancer cell lines. We went further and evaluated the role of a commercial FAK tyrosine kinase inhibitor VS-4718 on the viability and radiosensitization of the pancreatic cell lines as well as its effect on the extracellular matrix (ECM) production from the pancreatic stellate cells. Furthermore, we tested the effect of combining radiation with VS-4718 in a three-dimensional (3D) multicellular pancreatic tumor spheroid model. RESULTS: A database analysis revealed a relevant increase in genetic alterations and mRNA expression of the PTK2 in PDAC, which were associated with lower progression-free survival. In vitro, there was only variation in the basal phosphorylation level of FAK in cell lines. VS-4718 radiosensitized pancreatic cell lines only in the presence of ECM-producing pancreatic stellate cells and markedly reduced the ECM production in the stromal cells. Finally, using a 3D multicellular tumor model, the combination of VS-4718 and radiotherapy significantly reduced the growth of tumor aggregates. CONCLUSION: Pharmacological inhibition of FAK in pancreatic cancer could be a novel therapeutic strategy as our results show a radiosensitization effect of VS-4718 in vitro in a multicellular 2D- and in a 3D-model of pancreatic cancer.