Human Pancreatic Stellate Cells Research Articles

Abstract 3714: Role of miR-29a in pancreatic stellate cell mediated stromal remodeling in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive form of malignancy with nearly equal incidence and mortality rates in patients. The disease is characterized by dense desmoplastic tumor-associated stroma, which prevents effective therapeutic delivery to the tumor core and confers resistance to chemotherapy. The central effectors of such characteristic stroma in PDAC are the pancreatic stellate cells (PSCs), which are activated by autocrine and paracrine signaling of the tumor cells and deposit excessive amounts of extracellular matrix (ECM) proteins- the major component of the stroma. This interaction of the PSCs with the cancer cells greatly promote tumor progression and metastasis. In our previous work, we demonstrated that microRNA (miRNA) 29a plays a key role in PSC-mediated stromal reaction, and that the loss of the molecule in PSCs accelerates ECM protein accumulation and cancer growth. To understand the mechanistic regulation of miR-29a in PDAC stroma, we performed RNA-seq analysis with human PSCs (hPSCs), which identified 197 genes that differentially expressed with miR-29a overexpression at a false discovery rate less than 0.05. This included 19 downregulated and one upregulated direct miR-29a targets. Of these, expression of six key targets with the highest fold changes, namely, ADAMTS2, IGF1, E2F7, LAMC1, CLDN1 and ITGA6 by miR-29a were validated at the protein level in hPSCs. In addition, canonical pathways associated with the target genes and their network interactions were assessed, which identified the association of the key targets with pathways such as collagen synthesis, insulin signaling, TGF-β and PI3K/Akt pathways. ADAMTS2, E2F7, LAMC1 and CLDN1 expressions were significantly upregulated in PDAC patient pancreas compared to that in healthy individuals. Together, the current study presents a comprehensive understanding of miR-29a mediation in stromal remodeling, and elucidates associated pathways in regulating PSCs' function in PDAC desmoplasia and pancreatic carcinogenesis. Citation Format: Shatovisha Dey, Sheng Liu, Tricia D. Factora, Solaema Taleb, Primavera Riverahernandez, Lata Udari, Xiaoling Zhong, Jun Wan, Janaiah Kota. Role of miR-29a in pancreatic stellate cell mediated stromal remodeling in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3714.

Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer.

Background: The tumor microenvironment (TME) is composed of fibro-inflammatory cells and extracellular matrix (ECM) components. However, the exact contribution of the various TME compartments towards therapeutic response is unknown. Here, we aim to dissect the specific contribution of tumor-associated macrophages (TAMs) towards drug delivery and response in pancreatic ductal adenocarcinoma (PDAC). Methods: The effect of gemcitabine was assessed in human and murine macrophages, human pancreatic stellate cells (hPSCs), and tumor cells (L3.6pl, BxPC3 and KPC) in vitro. The drug metabolism of gemcitabine was analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Preclinical studies were conducted using KrasG12D;p48-Cre and KrasG12D;p53172H;Pdx-Cre mice to investigate gemcitabine delivery at different stages of tumor progression and upon pharmacological TAM depletion. Results: Gemcitabine accumulation was significantly increased in murine PDAC tissue compared to pancreatic intraepithelial neoplasia (PanIN) lesions and healthy control pancreas tissue. In vitro, macrophages accumulated and rapidly metabolized gemcitabine resulting in a significant drug scavenging effect for gemcitabine. Finally, pharmacological TAM depletion enhanced therapeutic response to gemcitabine in tumor-bearing KPC mice. Conclusion: Macrophages rapidly metabolize gemcitabine in vitro, and pharmacological depletion improves the therapeutic response to gemcitabine in vivo. Our study supports the notion that TAMs might be a promising therapeutic target in PDAC.

Isoliquiritigenin ameliorates caerulein-induced chronic pancreatitis by inhibiting the activation of PSCs and pancreatic infiltration of macrophages.

Chronic pancreatitis (CP) is characterized by persistent inflammation of the pancreas that results in progressive loss of the endocrine and exocrine compartment owing to atrophy and/or replacement with fibrotic tissue. Currently, the clinical therapeutic scheme of CP is mainly symptomatic treatment including pancreatic enzyme replacement, glycaemic control and nutritional support therapy, lacking of specific therapeutic drugs for prevention and suppression of inflammation and fibrosis aggravating in CP. Here, we investigated the effect of isoliquiritigenin (ILG), a chalcone‐type dietary compound derived from licorice, on pancreatic fibrosis and inflammation in a model of caerulein‐induced murine CP, and the results indicated that ILG notably alleviated pancreatic fibrosis and infiltration of macrophages. Further in vitro studies in human pancreatic stellate cells (hPSCs) showed that ILG exerted significant inhibition on the proliferation and activation of hPSCs, which may be due to negative regulation of the ERK1/2 and JNK1/2 activities. Moreover, ILG significantly restrained the M1 polarization of macrophages (RAW 264.7) via attenuation of the NF‐κB signalling pathway, whereas the M2 polarization was hardly affected. These findings indicated that ILG might be a potential anti‐inflammatory and anti‐fibrotic therapeutic agent for CP.

Targeting the CBP/β-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

Background: Although cyclic AMP-response element binding protein-binding protein (CBP)/β-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. Methods: To investigate the role of the CBP/β-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/β-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. Results: We report for the first time that CBP/β-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of “activation” markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/β-catenin antagonism also suppresses PSC-induced migration of cancer cells. Conclusion: CBP/β-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model

BackgroundPancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells.MethodA spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting.ResultsWe found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids.ConclusionsWe developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Metabolic regulation of calcium pumps in pancreatic cancer: role of phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3)

BackgroundHigh glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells.MethodsPDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADP-ribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging.ResultsPFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2.ConclusionsPFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.

Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumour growth and eliminates metastasis

BackgroundStromal–tumour interactions facilitate pancreatic cancer (PC) progression. The hepatocyte growth factor (HGF)/c-MET pathway is upregulated in PC and mediates the interaction between cancer cells and stromal pancreatic stellate cells (PSCs). This study assessed the effect of HGF/c-MET inhibition plus gemcitabine (G) on the progression of advanced PC.MethodsOrthotopic PC was produced by implantation of luciferase-tagged human cancer cells + human PSCs into mouse pancreas. Tumours were allowed to develop without treatment for 4 weeks. Mice were then treated for 6 weeks with one of the following: IgG, G, HGF inhibitor (Hi), c-MET inhibitor (Ci), Hi + Ci, Hi + G, Ci + G, or Hi + Ci + G.ResultsBioluminescence imaging showed similar tumour sizes in all mice at the initiation of treatments. Triple therapy (Hi + Ci + G): (1) completely eliminated metastasis; (2) significantly reduced tumour size as assessed by bioluminescence and at necropsy; (3) significantly reduced proliferating cancer cell density and stem cell marker DCLK1 expression in tumours. In vitro 3D culture studies supported our in vivo findings.ConclusionEven at an advanced disease stage, a two-pronged approach, targeting (a) HGF/c-MET with relevant inhibitors and (b) cancer cells with chemotherapy, completely eliminated metastasis and significantly decreased tumour growth, suggesting that this is a promising treatment approach for PC.

A Novel Way of Preventing Postoperative Pancreatic Fistula by Directly Injecting Profibrogenic Materials into the Pancreatic Parenchyma.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-β1 (TGF-β1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-β1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-β1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.

Establishment and Characterization of Paired Primary Cultures of Human Pancreatic Cancer Cells and Stellate Cells Derived from the Same Tumor.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis, and its treatment remains a challenge. As the existing in vitro experimental models offer only a limited resemblance to human PDAC, there is a strong need for additional research tools to better understand PDAC tumor biology, particularly the impact of the tumor stroma. Here, we report for the first time the establishment and characterization of human PDAC-derived paired primary monolayer cultures of (epithelial) cancer cells (PCCs) and mesenchymal stellate cells (PSCs) derived from the same tumor by the outgrowth method. Characterization of cell morphology, cytostructural, and functional profiles and proteomics-based secretome analysis were performed. All PCCs harbored KRAS and TP53 mutations, and expressed cytokeratin 19, ki-67, and p53, while the expression of EpCAM and vimentin was variable. All PSCs expressed α-smooth muscle actin (α-SMA) and vimentin. PCCs showed a significantly higher growth rate and proliferation than PSCs. Secretome analysis confirmed the distinct nature of PCCs as compared to PSCs and allowed identification of potential molecular regulators of PSC-conditioned medium (PSC-CM)-induced migration of PCCs. Paired primary cultures of PCCs and PSCs derived from the same tumor specimen represent a novel experimental model for basic research in PDAC tumor biology.

Nicotine promotes activation of human pancreatic stellate cells through inducing autophagy via α7nAChR-mediated JAK2/STAT3 signaling pathway

AimPancreatic stellate cells (PSCs) are the main functional cells leading to pancreatic fibrosis. Nicotine is widely considered as an independent risk factor of pancreatic fibrosis, but the mechanism is still unclear. Our study was aimed to explore the effects of nicotine on human pancreatic stellate cells (hPSCs) and involved pathways. Materials and methodsPrimary human PSCs were cultured and treated with nicotine (0.1 μM and 1 μM). The proliferation, apoptosis, α-SMA expression, extracellular matrix metabolism and autophagy of hPSCs were detected by CCK-8 assay, flow cytometry, real-time PCR and Western blotting analysis. The α7nAChR-mediated JAK2/STAT3 signaling pathway was also examined, and an α7nAChR antagonist α-bungarotoxin (α-BTX) was used to perform inhibition experiments. Key findingsThe proliferation, α-SMA expression and autophagy of hPSCs were significantly promoted by 1 μM nicotine. Meanwhile, the apoptosis of hPSCs was significantly reduced. The extracellular matrix metabolism of hPSCs was also regulated by nicotine. Moreover, the α7nAChR-mediated JAK2/STAT3 signaling pathway was activated by nicotine, this pathway and effects of nicotine can be blocked by α-BTX. SignificanceOur finding suggests that nicotine can promote activation of human pancreatic stellate cells (hPSCs) through inducing autophagy via α7nAChR-mediated JAK2/STAT3 signaling pathway, providing a new insight into the mechanisms by which nicotine affects pancreatic fibrosis.

The convergence of fibrosis and immune pathways: 5-HT2 and 5-HT2B antagonists attenuate profibrotic phenotype in human pancreatic stellate cells by modulating signal transducer and activator of transcription 3 phosphorylation

Background: Immune mechanisms are believed to play an important role in the pathogenesis of pancreatic inflammation and fibrosis. Transforming growth factor β1-mediated mechanisms are well known; however, an effective treatment option is yet not available. Serotonin (5-HT) is known to induce fibrosis in carcinoid tumors and diseases such as scleroderma and blocking 5-HT has shown promise in the attenuation of fibrosis in animal models of scleroderma. Herein, we evaluated the role of 5-HT in pancreatic fibrosis and its reversal with antagonists of 5-HT. Methodology: Pancreatic stellate cells (PSCs) were cultured from the two patients of pancreatic cancer undergoing pancreaticoduodenectomy. PSCs were cultured in the presence of 5-HT and as per the disease mimicking strategy (PSCs cultured with 5-HT first followed by 5-HT antagonist (terguride) and 5-HT2Bantagonist (SB204741) for 24 h and pretreatment strategy, PSCs treated with 5-HT antagonists first followed by 5-HT. mRNA expression of profibrotic genes (col1A1, Col1A2, ACTA-2, and CTGF) and Western blot analysis of signal transducer and activator of transcription 3 (STAT-3) and Smad-3 proteins was carried out. Results: 5-HT-induced profibrotic genes mRNA expression in PSCs was significantly reversed by 5-HT antagonists, terguride and SB204741, to a large extent. Pretreatment strategy was more effective in the amelioration of profibrotic potential. SB204741, 5-HT2Bantagonist was more effective than terguride. 5-HT antagonists mediated their effect by reducing the phosphorylation of noncanonical pathway mediator pSTAT3 more significantly then canonical pathway mediator pSmad-3. Conclusion: 5-HT induces profibrotic potential in human PSCs and inhibitors of 5-HT receptors, Terguride and SB 204,741 ameliorate it by reducing the phosphorylation of STAT-3 and Smad-3, more so in pretreatment strategy. 5-HT inhibitors may have a potential to ameliorate fibrosis in pancreas.

Biological and Proteomic Characteristics of an Immortalized Human Pancreatic Stellate Cell Line.

Human pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during chronic pancreatitis (CP). However, primary PSCs have a short lifespan in vitro, which seriously affects their use in various applications. We have established a stable immortalized human PSC line (HP-1) by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated human PSCs. HP-1 cells express cytoskeleton proteins including glial fibrillary acidic protein (GFAP), α-smooth muscle actin (α-SMA), vimentin and desmin, and are typical of PSCs, which are high transfeciability and viable in 0.5% serum. The cells express receptors such as TGFβR2, PDGFR, TGF-β pseudoreceptor Bambi and PPRPγ that are commonly found in PSCs. HP-1 cells are similar to activated human PSCs in that they have enhanced expression of α-SMA, CTGF, Col1 and TIMP-2 mRNAs or proteins, as well as decreased expression of MMP-1/2 mRNAs or proteins in response to TGF-β1 stimulation. Comparative proteomics revealed 4,537 shared proteins between HP-1 cells and PSCs and no single protein in HP-1 cells versus PSCs. Statistical analysis reveals no significantly difference between HP-1 cells and PSCs in their expression of proteins associated with matrix and matrix remodeling. The similarity between HP-1 cell and PSC is further shown by the finding that only 9 proteins are differentially up-regulated > ± 2-fold in HP-1 cells and 13 proteins are up-regulated > ± 2-fold in PSCs and none of these proteins include ECM proteins, cytokines, growth factors or matrix remodeling regulatory proteins. Therefore, HP-1 cells can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.

Resveratrol Ameliorates the Malignant Progression of Pancreatic Cancer by Inhibiting Hypoxia-induced Pancreatic Stellate Cell Activation

Pancreatic cancer is characterized by a hypoxic tumor microenvironment, which is primarily caused by massive fibrosis with pancreatic stellate cells (PSCs) as a main component. Our previous studies have shown that resveratrol can significantly inhibit pancreatic cancer. However, whether resveratrol can inhibit hypoxia-induced cancer development remains unclear. The objective of this study was to explore whether PSCs and hypoxia synergistically mediate aggressiveness in pancreatic cancer and detect the potential pleiotropic protective effects of resveratrol on hypoxia-induced pancreatic cancer progression. Human PSCs were treated with vehicle or resveratrol under normoxic or hypoxic conditions (3% O2), and PSC activation was assessed by immunofluorescence staining. SiRNA was used to silence hypoxia-inducible factor 1 (HIF-1) expression. The invasive capacity of Panc-1 and Mia Paca-2 cells cocultured with conditioned medium from PSCs was assessed by Transwell assays. To examine tumor formation kinetics, KPC (LSL-KrasG12D/+, Trp53fl/+, and Pdx1-Cre) mice were sacrificed at different time points. To investigate the antitumor effects of resveratrol in vivo, 8-wk-old KPC mice were divided into two groups and treated daily with or without 50 mg/kg resveratrol. Our data indicate that hypoxia induces PSC activation via HIF-1 and that the interleukin 6, vascular endothelial growth factor A, and stromal cell-derived factor 1 derived from activated PSCs promote both invasion and the epithelial–mesenchymal transition and inhibit apoptosis in pancreatic cancer cells. However, resveratrol inhibits hypoxia-induced PSC activation, blocks the interplay between PSCs and pancreatic cancer cells, and suppresses the malignant progression of pancreatic cancer and stromal desmoplasia in a KPC mouse model. Our data highlight that activated PSCs and intratumoral hypoxia are essential targets for novel strategies to prevent tumor–microenvironment interactions. Furthermore, the polyphenolic compound resveratrol effectively ameliorates the malignant progression of pancreatic ductal adenocarcinoma.

FGF2 engineered SPIONs attenuate tumor stroma and potentiate the effect of chemotherapy in 3D heterospheroidal model of pancreatic tumor.

Pancreatic ductal adenocarcinoma (PDAC), characterized with abundant tumor stroma, is a highly malignant tumor with poor prognosis. The tumor stroma largely consists of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), and is known to promote tumor growth and progression as well as acts as a barrier to chemotherapy. Inhibition of tumor stroma is highly crucial to induce the effect of chemotherapy. In this study, we delivered fibroblast growth factor 2 (FGF2) to human pancreatic stellate cells (hPSCs), the precursors of CAFs, using superparamagnetic iron oxide nanoparticles (SPIONs). FGF2 was covalently conjugated to functionalized PEGylated dextran-coated SPIONs. FGF2-SPIONs significantly reduced TGF-β induced hPSCs differentiation (α-SMA and collagen-1 expression) by inhibiting pSmad2/3 signaling and inducing ERK1/2 activity, as shown with western blot analysis. Then, we established a stroma-rich self-assembling 3D heterospheroid model by co-culturing PANC-1 and hPSCs in 3D environment. We found that FGF2-SPIONs treatment alone inhibited the tumor stroma-induced spheroid growth. In addition, they also potentiated the effect of gemcitabine, as shown by measuring the spheroid size and ATP content. These effects were attributed to the reduced expression of the hPSC activation and differentiation marker, α-SMA. Furthermore, to demonstrate an application of SPIONs, we applied an external magnetic field to spheroids while incubated with FGF2-SPIONs. This resulted in an enhanced effect of gemcitabine in our 3D model. In conclusion, this study presents a novel approach to target FGF2 to tumor stroma using SPIONs and thereby enhancing the effect of gemcitabine as demonstrated in the complex 3D tumor spheroid model.

Abstract A55: Pancreatic stellate cells promote pancreatic cancer invasion and metastasis by secretion of soluble factors and through contact-mediated mechanisms

Abstract Pancreatic ductal adenocarcinoma (PDAC) is rarely detected early, instead presenting in patients as advanced metastatic disease once symptoms are evident. Given the central role of metastasis in pancreatic tumor progression and mortality, there is an urgent need for further investigation of how PDAC metastasizes and for novel treatment strategies targeting PDAC metastasis. A prominent histopathologic feature of PDAC is a desmoplastic, fibrotic stroma consisting of extracellular matrix components, immune cells, and activated fibroblasts termed “pancreatic stellate cells” (PSCs) that often compose a majority of cellular content in many PDAC tumors. Data from recent studies demonstrate multiple phenotypic PSC populations in the stroma (Ohlund et al., 2017); however, the influence of individual PSC populations on metastasis and disease progression is not fully understood. We hypothesized that PSCs promote pancreatic tumor metastasis through secretion of soluble factors and accompany these metastasizing tumor cells to distal sites using collective invasion mechanisms. Tumor spheroids were formed by centrifugation of HPAC cells (a human PDAC cell line) in low adhesion round bottom plates. Spheroids were embedded in both collagen- and Matrigel-based matrices and z-stacks were collected every 24hrs for 72hrs using a Lecia SP8 confocal microscope. Live cell imaging was performed in an incubation chamber for 72hrs using a Leica SP8 confocal microscope. Human pancreatic stellate cells were isolated from surgically resected tumor specimens at Emory University Hospital under IRB-approved protocols and grown to ~70% confluency before supernatants were collected. Utilizing innovative 3D invasion assays, we demonstrate the ability of human PSC conditioned media to initiate and promote invasion of HPAC pancreatic tumor spheroids. HPAC spheroids exposed to 10% PSC supernatants showed significantly decreased roundness in comparison to media alone. Further investigation of possible soluble factors mediating this interaction indicated a protein-based interaction, although alternative mechanisms by which metastasis may be initiated were also identified. Phenotypic analysis of invasion through live cell imaging led to observations of unique invasion mechanisms by which hyperactive cells at the leading edge seemingly guide invasion of large groups of cells. Additionally, we observed co-culture interactions between PSCs and HPAC cells. In comparison to HPAC cells alone, ratios of 10:1 PSCs to HPAC cells increased circularity (less invasive) while inverse ratios decreased circularity of spheroids (more invasive). This study supports the hypothesis that PSCs can promote collective invasion and migration of pancreatic tumor cells through both contact-dependent and -independent means. Further identification of mechanisms mediating this interaction may reveal novel therapeutic targets for the treatment of PDAC and present new strategies for targeting metastasis of this deadly disease. Citation Format: Michael B. Ware, Jerry Yue, Bassel El-Rayes, Shishir K. Maithel, Juan Sarmiento, Gregory B. Lesinski. Pancreatic stellate cells promote pancreatic cancer invasion and metastasis by secretion of soluble factors and through contact-mediated mechanisms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A55.

Abstract A16: A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma

Abstract Background: Pancreatic cancer (PaCa) is a leading cause of cancer-related deaths and is projected to be the second most common cancer in a decade. It is characterized by a dense stroma surrounding tumor cells, known as desmoplasia that is stimulated by Sonic Hedgehog (SHH) released by PaCa cells. Stromal cells, in turn, release mitogenic substances such as hepatocyte growth factor (HGF) and Insulin-like growth factor receptor (IGF-1) by glioma-associated oncogene homolog 1 (GLI1) transcription factor that stimulate tumor growth, invasion, and resistance to therapy. We investigated the potential of targeting the metastasis suppressor gene N-myc downstream regulated gene 1 (NDRG1) to interrupt the oncogenic crosstalk between cancer associated human pancreatic stellate cells (PSCs) and PaCa cells. Methods: We assessed novel thiosemicarbazone anti-cancer agents, Dp44mT and DpC, that upregulate NDRG1 and their effects on: (i) SHH production by PaCa cells and also HGF and IGF-1 production by PSCs; (ii) HGF and IGF-1 receptors and their downstream signaling in indirect 2D and direct 3D co-culture of PSCs and PaCa cells; (iii) GLI1 and its upstream mediators in PSCs; and (iv) proliferative and metastatic properties of PaCa cells co-cultured with PSCs. Results: Dp44mT and DpC upregulated NDRG1 and reduced SHH production by PaCa. These agents also inhibited downstream HGF and IGF-1 signaling pathways in PaCa cells, leading to reduced metastatic potential in co-culture 2D and 3D models. Further, these agents also inhibited GLI1 levels and activation in PSCs, leading to reduced HGF and IGF-1 production by these stromal cells. NDRG1 overexpression alone had similar effects, while silencing NDRG1 further potentiated the oncogenic crosstalk between PaCa and PSC cells. Conclusions: Novel agents targeting NDRG1 have the unique ability to block the oncogenic crosstalk between PaCa cells and the surrounding stroma. This presents a novel approach to PaCa treatment that can potentially overcome the major problems attributed to desmoplasia, including therapeutic resistance and metastasis. Citation Format: Bekesho Geleta, Kyung Chan Park, Patric J. Jansson, Minoti Apte, Des R. Richardson, Zaklina Kovacevic. A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A16.

Yes-Associated Protein 1 Plays Major Roles in Pancreatic Stellate Cell Activation and Fibroinflammatory Responses.

Background: Yes-associated protein 1 (YAP), a transcriptional co-activator and major effector of the Hippo pathway, regulates cell differentiation and morphology in many cell types and supports aberrant tumor growth. Recent studies showed that YAP is expressed in pancreas tissues in pancreatic ductal adenocarcinoma (PDAC) patients and experimental models of PDAC, with YAP largely found in cancer cells and pancreatic stellate cells (PaSC) in the stroma.Methods and Results: We studied here the role of YAP in the activated phenotype of PaSC. We found that YAP is expressed at low levels in normal mouse pancreas, but protein levels significantly increased after pancreas inflammatory damage induced by repeated cerulein administration in wild-type mice or upon initiation of neoplastic transformation of the pancreas parenchyma in Ptf1-Cre;LSL-KrasG12D/+ (KC) mice. In these animal models, YAP upregulation occurred in parallel with activation and proliferation of PaSC. Consistent with these findings, we found robust YAP expression in culture-activated mouse and human PaSC but not in quiescent, freshly isolated cells. Fully activated PaSC isolated from KC mice or PDAC patient tissues exhibited robust nuclear YAP suggesting YAP transcriptional activity. Agents that induce quiescence such as the Bromodomain and Extra-Terminal (BET) inhibitor iBET151 and the p38 MAPK inhibitor SB203580 reduced YAP levels in PaSC. Stimulation of PaSC with the potent mitogen PDGF elicited marked YAP Ser127 phosphorylation. However, unexpectedly, this effect did not diminish YAP nuclear localization, suggesting that YAP phosphorylation at this site does not govern YAP cellular localization in PaSC. siRNA-mediated knockdown of YAP reduced PDGF-induced PaSC expansion in culture and blunted the persistent activation of Akt and ERK elicited by PDGF stimulation, supporting a role for YAP in PDGF-induced cell growth. YAP knockdown also blunted fibroinflammatory gene expression responses both in unstimulated and transforming growth factor beta 1 (TGFβ1)-stimulated PaSC.Conclusion: Our data suggest a central role for YAP in sustaining the activated phenotype and fibroinflammatory responses in PaSC. Moreover, our findings indicate that a complex crosstalk between YAP, TGFβ1, and PDGF pathways regulates PaSC activity and growth.

Cancer Cell Membrane-Camouflaged Nanorods with Endoplasmic Reticulum Targeting for Improved Antitumor Therapy.

Cell membrane-coated nanocarriers have been developed for drug delivery due to their enhanced blood circulation and tissue targeting capacities; however, previous works have generally focused on spherical nanoparticles and extracellular barriers. Many living organisms with different shapes, such as rod-shaped bacilli and rhabdovirus, display different functionalities regarding tissue penetration, cellular uptake, and intracellular distribution. Herein, we developed cancer cell membrane (CCM)-coated nanoparticles with spherical and rod shapes. CCM-coated nanorods (CRs) showed superior endocytosis efficiency compared with their spherical counterparts (CCM-coated nanospheres, CSs) due to the caveolin-mediated pathway. Moreover, CRs can effectively accumulate in the endoplasmic reticulum (ER) region and ship the loaded DOX to the nucleus at a considerable concentration, resulting in ER stress and subsequent apoptosis. After intravenous injection into human pancreatic adenocarcinoma cell (BxPC-3) and pancreatic stellate cell (HPSC) hybrid tumor-bearing nude mice, CRs exhibited improved immune escape ability, rapid extracellular matrix (ECM) penetration (8.2-fold higher than CSs), and enhanced tumor accumulation, further contributing to the enhanced antitumor efficacy. These findings may actually suggest the significance of shape design in improving current cell membrane-based drug delivery systems for effective subcellular targets and tumor therapy.

Verteporfin- and sodium porfimer-mediated photodynamic therapy enhances pancreatic cancer cell death without activating stromal cells in the microenvironment.

.The goal of our study was to determine the susceptibility of different pancreatic cell lines to clinically applicable photodynamic therapy (PDT). The efficacy of PDT of two different commercially available photosensitizers, verteporfin and sodium porfimer, was compared using a panel of four different pancreatic cancer cell lines, PANC-1, BxPC-3, CAPAN-2, and MIA PaCa-2, and an immortalized non-neoplastic pancreatic ductal epithelium cell line, HPNE. The minimum effective concentrations and dose-dependent curves of verteporfin and sodium porfimer on PANC-1 were determined. Since pancreatic cancer is known to have significant stromal components, the effect of PDT on stromal cells was also assessed. To mimic tumor–stroma interaction, a co-culture of primary human fibroblasts or human pancreatic stellate cell (HPSCs) line with PANC-1 was used to test verteporfin-PDT-mediated cell death of PANC-1. Two cytokines (TNF- and IL-) were used for stimulation of primary fibroblasts (derived from human esophageal biopsies) or HPSCs. The increased expression of smooth muscle actin (-SMA) confirmed the activation of fibroblasts or HPSC upon treatment with TNF- and IL-. Cell death assays showed that both sodium porfimer- and verteporfin-mediated PDT-induced cell death in a dose-dependent manner. However, verteporfin-PDT treatment had a greater efficiency with lower concentration than sodium porfimer-PDT in the PANC-1 incubated with stimulated fibroblasts or HPSC. Moreover, activation of stromal cells did not affect the treatment of the pancreatic cancer cell lines, suggesting that the effects of PDT are independent of the inflammatory microenvironment found in this two-dimensional culture model of cancers.

Obestatin activates mitogen-activated protein kinases associated to the obestatin/G-protein coupled receptor 39 signaling pathway in human pancreatic stellate cells

Venous invasion is not included in the pancreatic ductal adenocarcinoma (PDAC) staging, and its correlation with prognosis remains unclear. We evaluated the prognostic impact of radiographic portal/superior mesenteric vein (PV/SMV) invasion, and its possibility of complementing T staging.We identified patients with non-metastatic PDAC using our institutional cohort, and divided them according to PV/SMV invasion at imaging, defined as >180-degree tumor-vessel interface or contour deformity. We conducted Cox proportional hazard regression, and compared survival in the original and 1:1 propensity score matched datasets.We identified 454 patients [PV/SMV(+): 172; PV/SMV(−): 282]. In the multivariate analysis, PV/SMV invasion, age (≥70 years), performance status, tumor size (2–4, >4 cm), lymph nodes >4, and arterial invasion was correlated with prognosis. The PV/SMV(+) group had a shorter overall survival (OS) than the PV/SMV(−) group in the original (14.4 vs. 20.9 months; P < 0.001) and matched datasets (14.3 vs. 17.2 months; P = 0.009). Among patients without arterial invasion (cT1–cT3), the PV/SMV(+) group had a shorter OS (15.9 vs. 21.2 months; P = 0.002). Moreover, their OS did not differ from that of patients with arterial invasion (cT4) (15.9 vs. 14.4 months; P = 0.907). Patients with vessel (artery/vein) invasion had a shorter OS than those without vessel invasion (14.5 vs. 21.2 months; P < 0.001).Radiographic PV/SMV invasion in non-metastatic PDAC was correlated with a poor prognosis. It could identify a group with shorter OS among patients without arterial invasion (cT1–cT3). It is suggested that inclusion of PV/SMV invasion in clinical T4 criteria should be considered.