Pancreatic Stellate Cells Cells Research Articles

Abstract B051: Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a 5-year survival rate of only 10%. One hallmark of PDAC’s tumor microenvironment is dense desmoplasia, due to abnormal accumulation of extracellular matrix and proliferative fibroblasts. During tumorigenesis, pancreatic stellate cells (PSCs) obtain myofibroblast-like signatures and contribute to the fibrotic environment of PDAC. Desmoplasia-induced hypovascularity severely limits oxygen and nutrient delivery. Our laboratory revealed that hypoxia is prominent even at the pancreatic intraepithelial neoplasia (PanIN) stage, a dominant precursor lesion of PDAC. Hypoxia inhibits oxygen-dependent metabolic processes. For example, unsaturated lipid biosynthesis relies on oxygen to maintain desaturase enzymatic activities. Hypoxia also stimulates lipid uptake. Previous studies demonstrated that RAS-transformed cells preferentially import unsaturated lysophosphatidylcholines (LPCs) from culture media. However, in a nutrient-limited fibrotic environment, the potential source of lipids is unclear. Interestingly, a lipidomic study of a PSC secretome displayed a significant amount of LPCs, suggesting that PSCs are the potential lipid source in the PDAC microenvironment. Our data indicate that exogenous unsaturated lipids sustain PDAC cell viability under hypoxia and nutrient deficiency. Moreover, we find PDAC cell survival under tumor-like stress is enhanced in PSC conditioned medium, but not in delipidated conditioned medium. Strikingly, we show that cancer-associated fibroblasts do not experience hypoxic stress as much as adjacent malignant epithelium cells in vivo, suggesting cancer-associated fibroblasts provide unsaturated (and other) lipids to PDAC cells. Based on our lipidomic mass spectra of PSC conditioned medium, we determined that LPCs are actively utilized by PDAC cells for phosphatidylcholine and triglyceride regeneration. By inhibiting LPC to PC conversion with the drug TSI-01, which targets LPCAT (acyltransferase), unsaturated LPC no longer sustains cancer cell survival and ER stress is reactivated. In this study, we demonstrated metabolic crosstalk between fibroblasts and PDAC cells under stress conditions. Our findings revealed a novel metabolic inhibitor to disrupt the metabolic crosstalk between PSC and pancreatic cancer cells which might benefit developing combinatorial therapy of PDAC. Citation Format: Xu Han, Michelle Burrows, Laura Kim, Clementina Mesaros, John Tobias, Brian Keith, Celeste Simon. Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B051.

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, TGFβ1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFβ1 treatment exerted a more substantial effect than TGFβ1 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.

Targeting Periostin Expression Makes Pancreatic Cancer Spheroids More Vulnerable to Natural Killer Cells

Pancreatic cancer (PaCa) characteristically has a dense tumor microenvironment, which results in poor patient prognosis. Pancreatic stellate cells (PSCs) are the most abundant cells in the PaCa microenvironment and the principal source of collagen. Periostin, a matricellular protein, is produced specifically by PSCs and promotes the aggressiveness of PaCa cells by facilitating extracellular collagen assembly. Here, we aimed to decrease extracellular collagen assembly by suppressing periostin, thereby increasing the cytotoxic activity of natural killer (NK) cells. Periostin expression was suppressed in PSCs (called PSC-P) using CRISPR-Cas9. PaCa cells (BxPC-3) were co-cultured with PSC and PSC-P cells in a 3D environment to form tumor spheroids mimicking the tumor microenvironment. The extracellular collagen production of spheroids was evaluated by Masson's trichrome staining. The cytotoxic activity of NK-92 cells was analyzed by flow cytometry and confocal microscopy via CD107a staining. Cell death in BxPC-3 cells was evaluated by measuring Annexin-V and PI positivity using flow cytometry. As a result, periostin suppression decreased extracellular collagen and increased the infiltration of NK-92 cells into spheroids, and induced cell death in PaCa cells. In conclusion, we suggest that periostin might be a therapeutic target for PaCa and further analysis is warranted using in vivo models for proof-of-concept.

Abstract 1565: Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer

Abstract Introduction: Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are the desmoplastic stroma that acts as a barrier to drug delivery and effector immune cell infiltration. In our efforts to identify mediators of therapeutic resistance, we identified the pro-inflammatory cytokine interleukin-1α (IL-1) to be critical in the activation and reprogramming of neighboring cancer-associated fibroblasts (CAFs) within the stroma to a pro-inflammatory phenotype to promote myeloid cell chemotaxis. Our mechanistic studies have identified the p38 MAPK pathway as a novel mediator of IL-1-induced CAF activation from quiescent stellate cells into the inflammatory fibroblast. Our central hypothesis is that disruption of the IL-1/p38 MAPK signaling cascade in pancreatic stellate cells (PSCs) and CAFs can improve therapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC tumors. Methods: Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with Pexmetinib and genetically with an shRNA lentiviral system in CAF and PSC cell lines. Inflammatory PSC/CAF activation was determined by qPCR, immunofluorescence, and vitamin A assay. Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the p38 inhibitor, pexmetinib (30mg/kg, daily PO), or vehicle control for 2.5 weeks prior to sacrifice. For survival studies, PKT mice were treated with vehicle, gemcitabine (20 μg/twice weekly), pexmetinib, or combination until moribund. Results: Inhibition of p38 MAPK in PSCs prevented activation into an inflammatory fibroblast in vitro when stimulated with IL-1 or tumor cell cocultures. p38 MAPK inhibition in vivo resulted in signification reduction of PDGFR+ CAFs in a PDAC GEMM. Additionally, p38 MAPK inhibition resulted in a significant reduction of circulating myeloid cells (CD11b+) and intratumoral monocytic MDSCs (Ly6C+). p38 inhibition, in combination with chemotherapy, significantly improves overall survival in a PDAC GEMM. Conclusions: These findings provide important mechanistic data to explore p38 MAPK inhibition to target the fibrotic stroma and reduce immunosuppressive myeloid levels in tumors and provide compelling preclinical evidence to combine pexmetinib with chemotherapy to improve overall survival in PDAC. Citation Format: Samara Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin Garrido, Nilesh Deshpande, Zhiqun Zhou, Jashodeep Datta, Nagaraj Nagathihalli, Nipun Merchant. Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1565.

Abstract PO-048: Lack of L1CAM increases tumorigenicity, stemness and tumor fibrosis in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of <8%, characterized by extensive fibrosis, which causes chemotherapy failure and tumor progression. Pancreatic stellate cells (PSCs) are the major cellular stromal component in PDAC, which contributes to extracellular matrix (ECM) deposition and tumor development. L1 cell adhesion molecule (L1CAM) expression is generally associated with metastases and poor prognosis in different human tumors, while its role in PDAC remains debated. We found that the majority of PDAC biopsies have reduced L1 expression that involves a more aggressive phenotype. We also found, in vitro, that the L1low cells had enhanced CSC features, including self-renewal, migration and chemoresistance. In vivo the L1low cells display augmented tumorigenic potential and were able to recapitulate the tumor heterogeneity compared to their L1high counterpart. Conversely, ectopic overexpression of L1 resulted in a consistent decrease in cell proliferation and reduction in the stem properties of tumor cells. This data indicates that restoration of L1 expression could counteracts the malignant behavior of the tumor cells, thus leading to a less aggressive phenotype. Mechanistically we found that the TGF-β1 secreted by the PSC increased in vitro the migratory potential and the resistance to gemcitabine of the PDAC cells through the reduction of L1 expression. PSC silenced for TGF-β1 were not able anymore to modulate the L1 expression, and in vivo when co-injected with PDAC cells do not sustain tumor formation as observed in the PSC control. The transcriptome profiling (by mRNA sequencing) of L1low cells revealed pathways associated with collagen secretion and degradation, suggesting that the absence of L1 in cancer cells concurs to the remodeling of the surrounding microenvironment. In vitro experiments with cancer cells knocked down for L1 showed an increase in collagen deposition, which contribute to enhance extracellular matrix (ECM) stiffness and tumor fibrosis. Interestingly, we demonstrated in co-culture experiments that the L1low PDAC cells stimulate the collagen production in PSC. By contrary, the overexpression of L1 has no significant effects on collagen production by PSC cells. Altogether these data demonstrate an intrigued cross-talk between PSC and PDAC cells, in which the PSCs represent a supportive niche for PDAC cells promoting their aggressiveness and stemness through the downregulation of L1CAM mediated by TGF-β1 and the secretion of collagen. Citation Format: Donatella Delle Cave, Silvia Buonaiuto, Vincenza Colonna, Enza Lonardo. Lack of L1CAM increases tumorigenicity, stemness and tumor fibrosis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-048.

Cannabinoids Inhibited Pancreatic Cancer via P-21 Activated Kinase 1 Mediated Pathway.

The anti-cancer effects of cannabinoids including CBD (Cannabidiol) and THC ((−)-trans-∆9-tetrahydrocannabinol) have been reported in the case of pancreatic cancer (PC). The connection of these cannabinoids to KRas oncogenes that mutate in more than 90% of PC, and their effects on PD-L1, a key target of immune checkpoint blockade, have not been thoroughly investigated. Using cell lines and mouse models of PC, the effects of CBD and THC on cancer growth, the interaction between PC cells and a stromal cell, namely pancreatic stellate cells (PSCs), and the mechanism(s) involved were determined by cell-based assays and mouse study in vivo. CBD and THC inhibited the proliferation of PC, PSC, and PSC-stimulated PC cells. They also suppressed pancreatic tumour growth in mice. Furthermore, CBD and/or THC reduced the expression of PD-L1 by either PC or PSC cells. Knockout of p-21 activated kinase 1 (PAK1, activated by KRas) in PC and PSC cells and, in mice, dramatically decreased or blocked these inhibitory effects of CBD and/or THC. These results indicated that CBD and THC exerted their inhibitions on PC and PSC via a p-21 activated kinase 1 (PAK1)-dependent pathway, suggesting that CBD and THC suppress Kras activated pathway by targeting PAK1. The inhibition by CBD and THC of PD-L1 expression will enhance the immune checkpoint blockade of PC.

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.

Cold Atmospheric Plasma-Treated PBS Eliminates Immunosuppressive Pancreatic Stellate Cells and Induces Immunogenic Cell Death of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a low response to treatment and a five-year survival rate below 5%. The ineffectiveness of treatment is partly because of an immunosuppressive tumor microenvironment, which comprises tumor-supportive pancreatic stellate cells (PSCs). Therefore, new therapeutic strategies are needed to tackle both the immunosuppressive PSC and pancreatic cancer cells (PCCs). Recently, physical cold atmospheric plasma consisting of reactive oxygen and nitrogen species has emerged as a novel treatment option for cancer. In this study, we investigated the cytotoxicity of plasma-treated phosphate-buffered saline (pPBS) using three PSC lines and four PCC lines and examined the immunogenicity of the induced cell death. We observed a decrease in the viability of PSC and PCC after pPBS treatment, with a higher efficacy in the latter. Two PCC lines expressed and released damage-associated molecular patterns characteristic of the induction of immunogenic cell death (ICD). In addition, pPBS-treated PCC were highly phagocytosed by dendritic cells (DCs), resulting in the maturation of DC. This indicates the high potential of pPBS to trigger ICD. In contrast, pPBS induced no ICD in PSC. In general, pPBS treatment of PCCs and PSCs created a more immunostimulatory secretion profile (higher TNF-α and IFN-γ, lower TGF-β) in coculture with DC. Altogether, these data show that plasma treatment via pPBS has the potential to induce ICD in PCCs and to reduce the immunosuppressive tumor microenvironment created by PSCs. Therefore, these data provide a strong experimental basis for further in vivo validation, which might potentially open the way for more successful combination strategies with immunotherapy for PDAC.

Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells.

Activated pancreatic stellate cells (PSCs) play a central role in the tumor stroma of pancreatic ductal adenocarcinoma (PDAC). Given the limited availability of patient-derived PSCs from PDAC, immortalized PSC cell lines of murine and human origin have been established; however, it is not elucidated whether differences in species, organ disease status, donor age, and immortalization alter the PSC phenotype and behavior compared to that of patient-derived primary PSC cultures. Therefore, a panel of commonly used PSC cultures was examined for important phenotypical and functional features: three primary cultures from human PDAC, one primary from normal human pancreas, and three immortalized (one from human, two from murine pancreas). Growth rate was considerably lower in primary PSCs from human PDAC. Basal collagen synthesis varied between the PSC cultures, and TGF-β stimulation increased collagen synthesis only in non-immortalized cultures. Differences in secretome composition were observed along with a divergence in the DNA synthesis, migration, and response to gemcitabine of PDAC cell lines that were grown in conditioned medium from the various PSC cultures. The findings reveal considerable differences in features and functions that are key to PSCs and in the interactions with PDAC. These observations may be relevant to researchers when selecting the most appropriate PSC culture for their experiments.

PO-278 Assessment of the influence of stellate cells on primary pancreatic cancer cell growth and drug resistance in a spheroid model: MET inhibitors to the rescue

IntroductionPancreatic ductal adenocarcinoma has one of the lowest 5 year survival rates among all cancers and it is going to be the 2nd leading cause of cancer death in 2030. Drug resistance and early metastasis are among the main causes of this dismal prognosis and tumour microenvironment may give a considerable contribution to this aggressive behaviour. Pancreatic stellate cells (PSCs) are the main source of cancer-associated fibroblasts in stroma, and are suspected to induce drug resistance by paracrine secretion of hepatocyte growth factor (HGF) and activation of the MET receptor in cancer cells.Material and methodsWe first examined the effect of human PSC conditioned medium on the growth and drug resistance of six different primary cell cultures isolated from PDAC patients by sulforhodamine B (SRB) assay growing as monolayers. Further, we developed a spheroid 3D-co-culture with PDAC5-SSEA4 and immortalised or primary PSC cells and examined the effects of different drugs by luciferase assay, immunofluoresence and confocal microscopy.Results and discussionsConditioned medium of stimulated PSC cells, i.e., primed with PDAC conditioned medium, gave growth advantage to different primary PDAC cells and made them several times more resistant to gemcitabine and oxaliplatin. PDAC5-SSEA4/PSC spheroids were much more resistant to gemcitabine and oxaliplatin compared to PDAC5-SSEA4 spheroids. However, MET inhibitors such as tivantinib and PHA-665752 were equally effective in homo and heterospheroids. Of note, immortalised and primary PSC cells had similar influences on the behaviour of PDAC cells in spheroids.ConclusionWe successfully developed a 3D-spheroid model to evaluate the interaction of primary PDAC cells with PSCs. Pharmacological studies provided evidence that spheroids containing PSCs are much more resistant to cytotoxic drugs. Conversely MET inhibitors seem to be valuable tools to overcome the drug resistance of PDAC cells caused by the presence of PSC cells.

Inactivation of human pancreatic ductal adenocarcinoma with atmospheric plasma treated media and water: a comparative study

In recent years, the interest in treating cancer cells with plasma treated media (PTM) and plasma treated water (PTW) has increased tremendously. However, the actions of PTM and PTW are still not entirely understood. For instance, it is not clear whether the action of PTM is due to a modification in proteins/amino acids after plasma treatment of the media, or due to reactive oxygen and nitrogen species (RONS) generated from the plasma, or a combination of both effects. To differentiate between the actions of RONS and modified proteins/amino acids on the treatment of cancer cells, we compared the effects of PTM and PTW on two different pancreatic ductal adenocarcinomas (MiaPaca-2, BxPc3) and pancreatic stellate cells (PSCs) (hPSC128-SV). PSCs closely interact with cancer cells to create a tumor-promoting environment that stimulates local tumor progression and metastasis. We treated culture media and deionized water with a cold atmospheric plasma (CAP) jet, and subsequently applied this PTM/PTW at various ratios to the pancreatic cancer and PSC cell lines. We evaluated cell death, intracellular ROS concentrations and the mRNA expression profiles of four oxidative stress-related genes, i.e. Mitogen-activated protein kinase 7 (MAPK7), B-cell lymphoma 2 (BCL2), Checkpoint kinase 1 (CHEK1) and DNA damage-inducible transcript 3, also known as C/EBP homologous protein (CHOP). Our findings demonstrate that PTM and PTW have a similar efficacy to kill pancreatic cancer cells, while PTW is slightly more effective in killing PSCs, as compared to PTM. Furthermore, we observed an enhancement of the intracellular ROS concentrations in both pancreatic cancer cells and PSCs. Thus, it is likely that under our experimental conditions, the anti-cancer activity of PTM can be attributed more to the RONS present in the treated liquid, than to the modification of proteins/amino acids in the media. Furthermore, the fact that the chemo-resistant PSCs were killed by PTM/PTW may offer possibilities for new anti-cancer therapies for pancreatic cancer cells, including PSCs.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

Immunogenic Potential Of Cold Atmospheric Plasma For The Treatment Of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) forms the third leading cause of cancer related deaths in western countries. PDAC is a tumor with a fibroblastic stroma compartment that consists of pancreatic stellate cells (PSC) which play a complex role in supporting carcinogenesis, immunosuppression and therapy resistance. Therefore, the 5-year survival rate for PDAC patients remains below a disappointing 8%, stressing the need for new and more effective treatments [1]. Recently, cold atmospheric plasma (CAP) has emerged as a potent treatment option for cancer [2]. Although, CAP is being investigated for several years [3], the involvement of the immune system after CAP treatment remains poorly understood. The immunogenic cell death (ICD) concept describes that the killing of cancer cells leads to direct activation of the immune system. Cancer cells dying in such an immunogenic fashion release so-called ‘danger-associated molecular patterns’ that are able to induce a specific antitumoral immune response. ICD can be elicited by several physical means such as irradiation and photodynamic therapy, providing a rationale for the induction of ICD after CAP treatment [4]. The aim of this study is to investigate the induction of a specific antitumoral immune response after CAP treatment in PDAC, in vitro. Phosphate-buffered saline (PBS) was treated with CAP, generated by the kINPenIND®, and subsequently added to the monocultures of both pancreatic cancer cell (PCC) lines and PSC lines. To evaluate the four most important hallmarks of ICD, being membrane exposure of calreticulin, secretion of ATP and release of HMGB1 and type I interferon, the treatment parameters were optimized (i.e. treatment time, gas flow and gap distance) to obtain 50% cell death. The cellular difference in sensitivity between these two cell types for CAP treatment was assessed through cytotoxic analysis. After attaining the optimal treatment parameters, we investigated the translocation of calreticulin onto the cell surface with flow cytometry. ATP secretion was investigated with a bioluminescence assay, while ELISA was used to monitor the release of HMGB1 and interferon type I in the extracellular compartment. Our data report a cytotoxic and immunogenic effect of CAP treatment in vitro on both PCC and PSC cell lines. These results warrant further in vivo validation to refine the involvement of the immune system after CAP treatment.

Abstract B031: Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell

Abstract Background: Better understanding of the molecular mechanisms of pancreatic adenocarcinoma (PDAC) metastasis is important for the development of effective treatments for this devastating disease. We have previously shown that overexpression of Semaphorin 3E (Sema3E) enhances PDAC cell growth and is associated with poor patient survival. Here, we further determined the effect of Sema3E on stromal cells and the molecular mechanisms contributing to PDAC metastasis. Materials and Methods: Panels of stable PDAC cell lines were generated using either lenti-Sema3E for overexpression or the Crispr/Cas9 system to knock out (KO) Sema3E in both panc28 and panc48 cell lines. The effects of Sema3E overexpression or KO were measured in orthotopically implanted xenograft tumor mouse models in respect to tumor size, metastasis, and survival. After coculture with pancreatic stellate cells (PSCs), the effects of Sema3E on PSC activation were evaluated by GFAP and αSMA staining. Mixtures of PDAC cell lines with PSCs in vivo tumor growth were also evaluated. Result: After orthotopically implanting a panel of Sema3E-overexpressing or knockout panc28 cell lines, we found that the survival span of the sema3E-overexpression cell implant group was significantly shorter than the vector control group (31±9 days vs. 51±15 days) (p<0.05). In addition, more lung metastases were found in the Panc28-Sema3E overexpressing group. Compared to the primary tumor, the lung metastases had increased sema3E expression. Furthermore, Sema3E overexpression promoted PSC activation with a greater ratio of αSMA to GFAP staining when cocultured with PSC cells. Cotransplant of Sema3E-overexpression cells and PSC cells promoted tumor growth and metastasis when compare to Sema3E-overexpressed cells alone. Conclusion: Sema3E overexpression in pancreatic cancer cells promotes tumor growth, lung metastasis, and poor survival. The interaction between Sema3E and PSC cells may play an important role in contributing to tumor growth and metastasis. Citation Format: Zhengdong Liang, Lin-Kin Yong, George Van Buren, William Fisher, Rosa Hwang, Changyi Chen, Qizhi Cathy Yao. Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B031.

Galectin-3 Mediates Tumor Cell–Stroma Interactions by Activating Pancreatic Stellate Cells to Produce Cytokines via Integrin Signaling

Pancreatic ductal adenocarcinoma (PDAC) is characterized by activated pancreatic stellate cells (PSCs), abundance of extracellular matrix (ECM), and production of cytokines and chemokines. Galectin 3 (GAL3), a β-galactoside-specific lectin, contributes to PDAC development but its effects on the stroma and cytokine production are unclear. The effect of recombinant human GAL3 (rGAL3) on activation of PSCs, production of cytokines, and ECM proteins was determined by proliferation, invasion, cytokine array, and quantitative polymerase chain reaction. We assessed co-cultures of PDAC cells with GAL3 genetic alterations with PSCs. Production of interleukin 8 (IL8) and activities of nuclear factor (NF)-κB were determined by enzyme-linked immunosorbent assay and luciferase reporter analyses. We studied the effects of inhibitors of NF-κB and integrin-linked kinase (ILK) on pathways activated by rGAL3. In analyses of the Gene Expression Omnibus database and our dataset, we observed higher levels of GAL3, IL8, and other cytokines in PDAC than in nontumor tissues. Production of IL8, granulocyte-macrophage colony-stimulating factor, chemokine ligand 1, and C-C motif chemokine ligand 2 increased in PSCs exposed to rGAL3 compared with controls. Culture of PSCs with PDAC cells that express different levels of GAL3 resulted in proliferation and invasion of PSCs that increased with level of GAL3. GAL3 stimulated transcription of IL8 through integrin subunit beta 1 (ITGB1) on PSCs, which activates NF-κB through ILK. Inhibitors of ILK or NF-κB or a neutralizing antibody against ITGB1 blocked transcription and production of IL8 from PSCs induced by rGAL3. The GAL3 inhibitor significantly reduced growth and metastases of orthotopic tumors that formed from PDAC and PSC cells co-implanted in mice. GAL3 activates PSC cells to produce inflammatory cytokines via ITGB1signaling to ILK and activation of NF-κB. Inhibition of this pathway reduced growth and metastases of pancreatic orthotopic tumors in mice.

Interleukin-15 stimulates natural killer cell-mediated killing of both human pancreatic cancer and stellate cells.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

Epithelial to Stromal Re-Distribution of Primary Cilia during Pancreatic Carcinogenesis.

BackgroundThe Hedgehog (HH) pathway is a mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that primary cilia (PC), the essential organelles for HH signal transduction, were lost in PDAC. The aim of this study was to determine the presence of PC in human normal pancreas, chronic pancreatitis, and during carcinogenesis to PDAC with focus on both epithelia and stroma.MethodsPC were analyzed in paraffin sections from normal pancreas, chronic pancreatitis, intraductal papillary-mucinous neoplasia, and PDAC, as well as in primary human pancreatic stellate cells (PSC) and pancreatic cancer cell lines by double immunofluorescence staining for acetylated α-tubuline and γ-tubuline. Co-staining for the HH receptors PTCH1, PTCH2 and SMO was also performed.ResultsPC are gradually lost during pancreatic carcinogenesis in the epithelium: the fraction of cells with PC gradually and significantly decreased from 32% in ducts of normal pancreas, to 21% in ducts of chronic pancreatitis, to 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and to 1.2% in invasive PDAC. However, this loss of PC in the neoplastic epithelium is accompanied by a gain of PC in the surrounding stroma. The fraction of stromal cells with PC significantly increased from 13% around normal ducts to about 30% around PanIN and PDAC. HH-receptors were detected in tumor stroma but not in epithelial cells. PC are also present in PSC and pancreatic cancer cell lines.ConclusionPC are not lost during pancreatic carcinogenesis but re-distributed from the epithelium to the stroma. This redistribution may explain the re-direction of HH signaling towards the stroma during pancreatic carcinogenesis.

Paracrine IL-6 signaling mediates the effects of pancreatic stellate cells on epithelial-mesenchymal transition via Stat3/Nrf2 pathway in pancreatic cancer cells

BackgroundWe previously showed that pancreatic stellate cells (PSC) secreted interleukin (IL)-6 and promoted pancreatic ductal adenocarcinoma (PDAC) cell proliferation via nuclear factor erythroid 2 (Nrf2)-mediated metabolic reprogramming. Epithelial-mesenchymal transition (EMT) is a key process for the metastatic cascade. To study the mechanism of PDAC progression to metastasis, we investigated the role of PSC-secreted IL-6 in activating EMT and the involvement of Nrf2 in this process. MethodsGene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion. ResultsPSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6. ConclusionsIL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway. General significanceTargeting Stat3/Nrf2 pathway activated by PSC-secreted IL-6 may provide a novel therapeutic option to improve the prognosis of PDAC.

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7‐/‐ animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours.

Inhibitors of Arg-Gly-Asp-Binding Integrins Reduce Development of Pancreatic Fibrosis in Mice.

Background & AimsPancreatic stellate cells (PSCs) regulate the development of chronic pancreatitis (CP) and are activated by the cytokine transforming growth factor β (TGFB). Integrins of the αv family promote TGFB signaling in mice, probably by interacting with the Arg-Gly-Asp (RGD) sequence of the TGFB latency-associated peptide, which frees TGFB to bind its cellular receptors. However, little is known about the role of integrins in the development of CP. We investigated the effects of small-molecule integrin inhibitors in a mouse model of CP.MethodsWe induced CP in C57BL/6 female mice by repeated cerulein administration. An active RGD peptidomimetic compound (Center for World Health and Medicine [CWHM]-12) was delivered by continuous infusion, starting 3 days before or 5 days after cerulein administration began. Pancreata were collected and parenchymal atrophy, fibrosis, and activation of PSCs were assessed by histologic, gene, and protein expression analyses. We measured CWHM-12 effects on activation of TGFB in co-culture assays in which rat PSC cells (large T immortalized cells [LTC-14]) activate expression of a TGFB-sensitive promoter in reporter cells.ResultsPancreatic tissues of mice expressed messenger RNAs encoding subunits of RGD-binding integrins. Cerulein administration increased expression of these integrins, altered pancreatic cell morphology, and induced fibrosis. The integrin inhibitor CWHM-12 decreased acinar cell atrophy and loss, and substantially reduced fibrosis, activation of PSCs, and expression of genes regulated by TGFB. CWHM-12 also reduced established fibrosis in mice and blocked activation of TGFB in cultured cells.ConclusionsBased on studies of a mouse model of CP and cultured PSCs, integrins that bind RGD sequences activate PSCs and promote the development of pancreatic fibrogenesis in mice. Small-molecule antagonists of this interaction might be developed for treatment of pancreatic fibrotic diseases.